Method of identifying nucleotide differences

ABSTRACT

The present invention provides methods and apparatus for sequencing, fingerprinting and mapping biological macromolecules, typically biological polymers. The methods make use of a plurality of sequence specific recognition reagents which can also be used for classification of biological samples, and to characterize their sources.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of application Ser. No. 08/670,118, filed Jun.25, 1996, now U.S. Pat. No. 5,800,992; which is a divisional ofapplication Ser. No. 08/168,904, filed Dec. 15, 1993, now abandoned;which is a continuation of application Ser. No. 07/624,114, filed Dec.6, 1990, now abandoned; which is a continuation in-part application ofcommonly assigned application Ser. No. 07/362,901, filed Jun. 7, 1989,now abandoned which are hereby incorporated by reference.

Additional commonly assigned application Ser. Nos. 07/624,120, nowabandoned and 07/626,730, now U.S. Pat. No. 5,356,779, both of whichwere filed on Dec. 6, 1990; application Ser. No. 07/435,316, filed Nov.13, 1989, now abandoned; and U.S. Pat. No. 5,252,743 are also herebyincorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates to the sequencing, fingerprinting, andmapping of polymers, particularly biological polymers. The inventionsmay be applied, for example, in the sequencing, fingerprinting, ormapping of nucleic acids, polypeptides, oligosaccharides, and syntheticpolymers.

The relationship between structure and function of macromolecules is offundamental importance in the understanding of biological systems. Theserelationships are important to understanding, for example, the functionsof enzymes, structural proteins, and signalling proteins, ways in whichcells communicate with each other, as well as mechanisms of cellularcontrol and metabolic feedback.

Genetic information is critical in continuation of life processes. Lifeis substantially informationally based and its genetic content controlsthe growth and reproduction of the organism and its complements.Polypeptides, which are critical features of all living systems, areencoded by the genetic material of the cell. In particular, theproperties of enzymes, functional proteins, and structural proteins aredetermined by the sequence of amino acids which make them up. Asstructure and function are integrally related, many biological functionsmay be explained by elucidating the underlying structural features whichprovide those functions. For this reason, it has become very importantto determine the genetic sequences of nucleotides which encode theenzymes, structural proteins, and other effectors of biologicalfunctions. In addition to segments of nucleotides which encodepolypeptides, there are many nucleotide sequences which are involved incontrol and regulation of gene expression.

The human genome project is directed toward determining the completesequence of the genome of the human organism. Although such a sequencewould not correspond to the sequence of any specific individual, itwould provide significant information as to the general organization andspecific sequences contained within segments from particularindividuals. It would also provide mapping information which is veryuseful for further detailed studies. However, the need for highly rapid,accurate, and inexpensive sequencing technology is nowhere more apparentthan in a demanding sequencing project such as this. To complete thesequencing of a human genome would require the determination ofapproximately 3×10⁹, or 3 billion base pairs.

The procedures typically used today for sequencing include the Sangerdideoxy method, see, e.g., Sanger et al. (1977) Proc. Natl. Acad. Sci.USA, 74:5463-5467, or the Maxam and Gilbert method, see, e.g., Maxam etal., (1980) Methods in Enzymology, 65:499-559. The Sanger methodutilizes enzymatic elongation procedures with chain terminatingnucleotides. The Maxam and Gilbert method uses chemical reactionsexhibiting specificity of reaction to generate nucleotide specificcleavages. Both methods require a practitioner to perform a large numberof complex manual manipulations. These manipulations usually requireisolating homogeneous DNA fragments, elaborate and tedious preparing ofsamples, preparing a separating gel, applying samples to the gel,electrophoresing the samples into this gel, working up the finished gel,and analyzing the results of the procedure.

Thus, a less expensive, highly reliable, and labor efficient means forsequencing biological macromolecules is needed. A substantial reductionin cost and increase in speed of nucleotide sequencing would be verymuch welcomed. In particular, an automated system would improve thereproducibility and accuracy of procedures. The present inventionsatisfies these and other needs.

SUMMARY OF THE INVENTION

The present invention provides improved methods useful for de novosequencing of an unknown polymer sequence, for verification of knownsequences, for fingerprinting polymers, and for mapping homologoussegments within a sequence. By reducing the number of manualmanipulations required and automating most of the steps, the speed,accuracy, and reliability of these procedures are greatly enhanced.

The production of a substrate having a matrix of positionally definedregions with attached reagents exhibiting known recognition specificitycan be used for the sequence analysis of a polymer. Although mostdirectly applicable to sequencing, the present invention is alsoapplicable to fingerprinting, mapping, and general screening of specificinteractions. The VLSIPS™ Technology (Very Large Scale ImmobilizedPolymer Synthesis) substrates will be applied to evaluating otherpolymers, e.g., carbohydrates, polypeptides, hydrocarbon syntheticpolymers, and the like. For these non-polynucleotides, the sequencespecific reagents will usually be antibodies specific for a particularsubunit sequence.

According to one aspect of the masking technique, the invention providesan ordered method for forming a plurality of polymer sequences bysequential addition of reagents comprising the step of seriallyprotecting and deprotecting portions of the plurality of polymersequences for addition of other portions of the polymer sequences usinga binary synthesis strategy.

The present invention also provides a means to automate sequencingmanipulations. The automation of the substrate production method and ofthe scan and analysis steps minimizes the need for human intervention.This simplifies the tasks and promotes reproducibility.

The present invention provides a composition comprising a plurality ofpositionally distinguishable sequence specific reagents attached to asolid substrate, which reagents are capable of specifically binding to apredetermined subunit sequence of a preselected multi-subunit lengthhaving at least three subunits, said reagents representing substantiallyall possible sequences of said preselected length. In some embodiments,the subunit sequence is a polynucleotide or a polypeptide, in others thepreselected multi-subunit length is five subunits and the subunitsequence is a polynucleotide sequence. In other embodiments, thespecific reagent is an oligonucleotide of at least about fivenucleotides. Alternatively, the specific reagent is a monoclonalantibody. Usually the specific reagents are all attached to a singlesolid substrate, and the reagents comprise about 3000 differentsequences. In other embodiments, the reagents represents at least about25% of the possible subsequences of said preselected length. Usually,the reagents are localized in regions of the substrate having a densityof at least 25 regions per square centimeter, and often the substratehas a surface area of less than about 4 square centimeters.

The present invention also provides methods for analyzing a sequence ofa polynucleotide or a polypeptide, said method comprising the step of:

a) exposing said polynucleotide or polypeptide to a composition asdescribed.

It also provides useful methods for identifying or comparing a targetsequence with a reference, said method comprising the step of:

a) exposing said target sequence to a composition as described;

b) determining the pattern of positions of the reagents whichspecifically interact with the target sequence; and

c) comparing the pattern with the pattern exhibited by the referencewhen exposed to the composition.

The present invention also provides methods for sequencing a segment ofa polynucleotide comprising the steps of:

a) combining:

i) a substrate comprising a plurality of chemically synthesized andpositionally distinguishable oligonucleotides capable of recognizingdefined oligonucleotide sequences; and

ii) a target polynucleotide; thereby forming high fidelity matchedduplex structures of complementary subsequences of known sequence; and

b) determining which of said reagents have specifically interacted withsubsequences in said target polynucleotide.

In one embodiment, the segment is substantially the entire length ofsaid polynucleotide.

The invention also provides methods for sequencing a polymer, saidmethod comprising the steps of:

a) preparing a plurality of reagents which each specifically bind to asubsequence of preselected length;

b) positionally attaching each of said reagents to one or more solidphase substrates, thereby producing substrates of positionally definablesequence specific probes;

c) combining said substrates with a target polymer whose sequence is tobe determined; and

d) determining which of said reagents have specifically interacted withsubsequences in said target polymer.

In one embodiment, the substrates are beads. Preferably, the pluralityof reagents comprise substantially all possible subsequences of saidpreselected length found in said target. In another embodiment, thesolid phase substrate is a single substrate having attached theretoreagents recognizing substantially all possible subsequences ofpreselected length found in said target.

In another embodiment, the method further comprises the step ofanalyzing a plurality of said recognized subsequences to assemble asequence of said target polymer. In a bead embodiment, at least some ofthe plurality of substrates have one subsequence specific reagentattached thereto, and the substrates are coded to indicate the sequencespecificity of said reagent.

The present invention also embraces a method of using a fluorescentnucleotide to detect interactions with oligonucleotide probes of knownsequence, said method comprising:

a) attaching said nucleotide to a target unknown polynucleotidesequence, and

b) exposing said target polynucleotide sequence to a collection ofpositionally defined oligonucleotide probes of known sequences todetermine the sequences of said probes which interact with said target.

In a further refinement, an additional step is included of:

a) collating said known sequences to determine the overlaps of saidknown sequences to determine the sequence of said target sequence.

A method of mapping a plurality of sequences relative to one another isalso provided, the method comprising:

a) preparing a substrate having a plurality of positionally attachedsequence specific probes;

b) exposing each of said sequences to said substrate, therebydetermining the patterns of interaction between said sequence specificprobes and said sequences; and

c) determining the relative locations of said sequence specific probeinteractions on said sequences to determine the overlaps and order ofsaid sequences.

In one refinement, the sequence specific probes are oligonucleotides,applicable to where the target sequences are nucleic acid sequences.

In the nucleic acid sequencing application, the steps of the sequencingprocess comprise:

a) producing a matrix substrate having known positionally definedregions of known sequence specific oligonucleotide probes;

b) hybridizing a target polynucleotide to the positions on the matrix sothat each of the positions which contain oligonucleotide probescomplementary to a sequence on the target hybridize to the targetmolecule;

c) detecting which positions have bound the target, thereby determiningsequences which are found on the target; and

d) analyzing the known sequences contained in the target to determinesequence overlaps and assembling the sequence of the target therefrom.

The enablement of the sequencing process by hybridization is based inlarge part upon the ability to synthesize a large number (e.g., tovirtually saturate) of the possible overlapping sequence segments anddistinguishing those probes which hybridize with fidelity from thosewhich have mismatched bases, and to analyze a highly complex pattern ofhybridization results to determine the overlap regions.

The detecting of the positions which bind the target sequence wouldtypically be through a fluorescent label on the target. Although afluorescent label is probably most convenient, other sorts of labels,e.g., radioactive, enzyme linked, optically detectable, or spectroscopiclabels may be used. Because the oligonucleotide probes are positionallydefined, the location of the hybridized duplex will directly translateto the sequences which hybridize. Thus, analysis of the positionsprovides a collection of subsequences found within the target sequence.These subsequences are matched with respect to their overlaps so as toassemble an intact target sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a flow chart for sequence, fingerprint, or mappinganalysis.

FIG. 2 illustrates the proper function of a VLSIPS™ Technologynucleotide synthesis.

FIG. 3 illustrates the proper function of a VLSIPS™ Technologynucleotide synthesis.

FIG. 4 illustrates the process of a VLSIPS™ Technology trinucleotidesynthesis.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Overall Description

A. general

B. VLSIPS substrates

C. binary masking

D. applications

E. detection methods and apparatus

F. data analysis

II. Theoretical Analysis

A. simple n-mer structure; theory

B. complications

C. non-polynucleotide embodiments

III. Polynucleotide Sequencing

A. preparation of substrate matrix

B. labeling target polynucleotide

C. hybridization conditions

D. detection; VLSIPS scanning

E. analysis

F. substrate reuse

G. non-polynucleotide aspects

IV. Fingerprinting

A. general

B. preparation of substrate matrix

C. labeling target nucleotides

D. hybridization conditions

E. detection; VLSIPS scanning

F. analysis

G. substrate reuse

H. non-polynucleotide aspects

V. Mapping

A. general

B. preparation of substrate matrix

C. labeling

D. hybridization/specific interaction

E. detection

F. analysis

G. substrate reuse

H. non-polynucleotide aspects

VI. Additional Screening

A. specific interactions

B. sequence comparisons

C. categorizations

D. statistical correlations

VII. Formation of Substrate

A. instrumentation

B. binary masking

C. synthetic methods

D. surface immobilization

VIII. Hybridization/Specific Interaction

A. general

B. important parameters

IX. Detection Methods

A. labeling techniques

B. scanning system

X. Data Analysis

A. general

B. hardware

C. software

XI. Substrate Reuse

A. removal of label

B. storage and preservation

C. processes to avoid degradation of oligomers

XII. Integrated Sequencing Strategy

A. initial mapping strategy

B. selection of smaller clones

C. actual sequencing procedures

XIII. Commercial Applications

A. sequencing

B. fingerprinting

C. mapping

I. OVERALL DESCRIPTION

A. General

The present invention relies in part on the ability to synthesize orattach specific recognition reagents at known locations on a substrate,typically a single substrate. In particular, the present inventionprovides the ability to prepare a substrate having a very high densitymatrix pattern of positionally defined specific recognition reagents.The reagents are capable of interacting with their specific targetswhile attached to the substrate, e.g., solid phase interactions, and byappropriate labeling of these targets, the sites of the interactionsbetween the target and the specific reagents may be derived. Because thereagents are positionally defined, the sites of the interactions willdefine the specificity of each interaction. As a result, a map of thepatterns of interactions with specific reagents on the substrate isconvertible into information on the specific interactions taking place,e.g., the recognized features. Where the specific reagents recognize alarge number of possible features, this system allows the determinationof the combination of specific interactions which exist on the targetmolecule. Where the number of features is sufficiently large, theidentical same combination, or pattern, of features is sufficientlyunlikely that a particular target molecule may often be uniquely definedby its features. In the extreme, the features may actually be thesubunit sequence of the target molecule, and a given target sequence maybe uniquely defined by its combination of features.

In particular, the methodology is applicable to sequencingpolynucleotides. The specific sequence recognition reagents willtypically be oligonucleotide probes which hybridize with specificity tosubsequences found on the target sequence. A sufficiently large numberof those probes allows the fingerprinting of a target polynucleotide orthe relative mapping of a collection of target polynucleotides, asdescribed in greater detail below.

In the high resolution fingerprinting provided by a saturatingcollection of probes which include all possible subsequences of a givensize, e.g., 10-mers, collating of all the subsequences and determinationof specific overlaps will be derived and the entire sequence can usuallybe reconstructed.

Although a polynucleotide sequence analysis is a preferred embodiment,for which the specific reagents are most easily accessible, theinvention is also applicable to analysis of other polymers, includingpolypeptides, carbohydrates, and synthetic polymers, including α-, β-,and ω-amino acids, polyurethanes, polyesters, polycarbonates, polyureas,polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes,polyimides, polyacetates, and mixed polymers. Various optical isomers,e.g., various D- and L- forms of the monomers, may be used.

Sequence analysis will take the form of complete sequence determination,to the level of the sequence of individual subunits along the entirelength of the target sequence. Sequence analysis also takes the form ofsequence homology, e.g., less than absolute subunit resolution, where"similarity" in the sequence will be detectable, or the form ofselective sequences of homology interspersed at specific or irregularlocations.

In either case, the sequence is determinable at selective resolution orat particular locations. Thus, the hybridization method will be usefulas a means for identification, e.g., a "fingerprint", much like aSouthern hybridization method is used. It is also useful to mapparticular target sequences.

B. VLSIPS™ Technology

The invention is enabled by the development of technology to preparesubstrates on which specific reagents may be either positionallyattached or synthesized. In particular, the very large scale immobilizedpolymer synthesis (VLSIPS™) technology allows for the very high densityproduction of an enormous diversity of reagents mapped out in a knownmatrix pattern on a substrate. These reagents specifically recognizesubsequences in a target polymer and bind thereto, producing a map ofpositionally defined regions of interaction. These map positions areconvertible into actual features recognized, and thus would be presentin the target molecule of interest.

As indicated, the sequence specific recognition reagents will often beoligonucleotides which hybridize with fidelity and discrimination to thetarget sequence. For use with other polymers, monoclonal or polyclonalantibodies having high sequence specificity will often be used.

In the generic sense, the VLSIPS technology allows the production of asubstrate with a high density matrix of positionally mapped regions withspecific recognition reagents attached at each distinct region. By useof protective groups which can be positionally removed, or added, theregions can be activated or deactivated for addition of particularreagents or compounds. Details of the protection are described below andin related Pirrung et al. (1992)U.S. Pat. No. 5,143,854. In a preferredembodiment, photosensitive protecting agents will be used and theregions of activation or deactivation may be controlled byelectro-optical and optical methods, similar to many of the processesused in semiconductor wafer and chip fabrication.

In the nucleic acid nucleotide sequencing application, a VLSIPSsubstrate is synthesized having positionally defined oligonucleotideprobes. See Pirrung et al. (1992) U.S. Pat. No. 5,143,854; and U.S. Ser.No. 07/624,120, now abandoned. By use of masking technology andphotosensitive synthetic subunits, the VLSIPS apparatus allows for thestepwise synthesis of polymers according to a positionally definedmatrix pattern. Each oligonucleotide probe will be synthesized at knownand defined positional locations on the substrate. This forms a matrixpattern of known relationship between position and specificity ofinteraction. The VLSIPS technology allows the production of a very largenumber of different oligonucleotide probes to be simultaneously andautomatically synthesized including numbers in excess of about 10², 10³,10⁴, 10⁵, 10⁶, or even more, and at densities of at least about 10², 10³/cm², 10⁴ /cm², 10⁵ /cm² and up to 10⁶ /cm² or more. This applicationdiscloses methods for synthesizing polymers on a silicon or othersuitably derivatized substrate, methods and chemistry for synthesizingspecific types of biological polymers on those substrates, apparatus forscanning and detecting whether interaction has occurred at specificlocations on the substrate, and various other technologies related tothe use of a high density very large scale immobilized polymersubstrate. In particular, sequencing, fingerprinting, and mappingapplications are discussed herein in detail, though related technologiesare described in simultaneously filed applications U.S. Ser. No.07/624,120, now abandoned; and U.S. Ser. No. 07/517,659; Dower et al.(1995) U.S. Pat. No. 5,427,908, each of which is hereby incorporatedherein by reference.

In other embodiments, antibody probes will be generated whichspecifically recognize particular subsequences found on a polymer.Antibodies would be generated which are specific for recognizing a threecontiguous amino acid sequence, and monoclonal antibodies may bepreferred. Optimally, these antibodies would not recognize any sequencesother than the specific three amino acid stretch desired and the bindingaffinity should be insensitive to flanking or remote sequences found ona target molecule. Likewise, antibodies specific for particularcarbohydrate linkages or sequences will be generated. A similar approachcould be used for preparing specific reagents which recognize otherpolymer subunit sequences. These reagents would typically be sitespecifically localized to a substrate matrix pattern where the regionsare closely packed.

These reagents could be individually attached at specific sites on thesubstrate in a matrix by an automated procedure where the regions arepositionally targeted by some other specific mechanism, e.g., one whichwould allow the entire collection of reagents to be attached to thesubstrate in a single reaction. Each reagent could be separatelyattached to a specific oligonucleotide sequence by an automatedprocedure. This would produce a collection of reagents where, e.g., eachmonoclonal antibody would have a unique oligonucleotide sequenceattached to it. By virtue of a VLSIPS substrate which has differentcomplementary oligonucleotides synthesized on it, each monoclonalantibody would specifically be bound only at that site on the substratewhere the complementary oligonucleotide has been synthesized. Acrosslinking step would fix the reagent to the substrate. See, e.g.,Dattagupta et al. (1985) U.S. Pat. No. 4,542,102 and (1987) U.S. Pat.No. 4,713,326; and Chatterjee, M. et al. (1990) J. Am. Chem. Soc.112:6397-6399, which are hereby incorporated herein by reference. Thisallows a high density positionally specific collection of specificrecognition reagents, e.g., monoclonal antibodies, to be immobilized toa solid substrate using an automated system.

The regions which define particular reagents will usually be generatedby selective protecting groups which may be activated or deactivated.Typically the protecting group will be bound to a monomer subunit orspatial region, and can be spatially affected by an activator, such aselectromagnetic radiation. Examples of protective groups with utilityherein include nitroveratryl oxycarbonyl (NVOC), nitrobenzyl oxycarbony(NBOC), dimethyl dimethoxy benzyloxy carbonyl, 5-bromo-7-nitroindolinyl,O-hydroxy-α-methyl cinnamoyl, and 2-oxymethylene anthraquinone. Examplesof activators include ion beams, electric fields, magnetic fields,electron beams, x-ray, and other forms of electromagnetic radiation.

C. Binary Masking

In fact, the means for producing a substrate useful for these techniquesare explained in Pirrung et al. (1992) U.S. Pat. No. 5,143,854, which ishereby incorporated herein by reference. However, there are variousparticular ways to optimize the synthetic processes. Many of thesemethods are described in Ser. No. 07/624,120, now abandoned.

Briefly, the binary synthesis strategy refers to an ordered strategy forparallel synthesis of diverse polymer sequences by sequential additionof reagents which may be represented by a reactant matrix, and a switchmatrix, the product of which is a product matrix. A reactant matrix is a1×n matrix of the building blocks to be added. The switch matrix is allor a subset of the binary numbers from 1 to n arranged in columns. Inpreferred embodiments, a binary strategy is one in which at least twosuccessive steps illuminate half of a region of interest on thesubstrate. In most preferred embodiments, binary synthesis refers to asynthesis strategy which also factors a previous addition step. Forexample, a strategy in which a switch matrix for a masking strategyhalves regions that were previously illuminated, illuminating about halfof the previously illuminated region and protecting the remaining half(while also protecting about half of previously protected regions andilluminating about half of previously protected regions). It will berecognized that binary rounds may be interspersed with non-binary roundsand that only a portion of a substrate may be subjected to a binaryscheme, but will still be considered to be a binary masking schemewithin the definition herein. A binary "masking" strategy is a binarysynthesis which uses light to remove protective groups from materialsfor addition of other materials such as nucleotides or amino acids.

In particular, this procedure provides a simplified and highly efficientmethod for saturating all possible sequences of a defined lengthpolymer. This masking strategy is also particularly useful in producingall possible oligonucleotide sequence probes of a given length.

D. Applications

The technology provided by the present invention has very broadapplications. Although described specifically for polynucleotidesequences, similar sequencing, fingerprinting, mapping, and screeningprocedures can be applied to polypeptide, carbohydrate, or otherpolymers. In particular, the present invention may be used to completelysequence a given target sequence to subunit resolution. This may be forde novo sequencing, or may be used in conjunction with a secondsequencing procedure to provide independent verification. See, e.g.,(1988) Science 242:1245. For example, a large polynucleotide sequencedefined by either the Maxam and Gilbert technique or by the Sangertechnique may be verified by using the present invention.

In addition, by selection of appropriate probes, a polynucleotidesequence can be fingerprinted. Fingerprinting is a less detailedsequence analysis which usually involves the characterization of asequence by a combination of defined features. Sequence fingerprintingis particularly useful because the repertoire of possible features whichcan be tested is virtually infinite. Moreover, the stringency ofmatching is also variable depending upon the application. A SouthernBlot analysis may be characterized as a means of simple fingerprintanalysis.

Fingerprinting analysis may be performed to the resolution of specificnucleotides, or may be used to determine homologies, most commonly forlarge segments. In particular, an array of oligonucleotide probes ofvirtually any workable size may be positionally localized on a matrixand used to probe a sequence for either absolute complementary matching,or homology to the desired level of stringency using selectedhybridization conditions.

In addition, the present invention provides means for mapping analysisof a target sequence or sequences. Mapping will usually involve thesequential ordering of a plurality of various sequences, or may involvethe localization of a particular sequence within a plurality ofsequences. This may be achieved by immobilizing particular largesegments onto the matrix and probing with a shorter sequence todetermine which of the large sequences contain that smaller sequence.Alternatively, relatively shorter probes of known or random sequence maybe immobilized to the matrix and a map of various different targetsequences may be determined from overlaps. Principles of such anapproach are described in some detail by Evans et al. (1989) "PhysicalMapping of Complex Genomes by Cosmid Multiplex Analysis," Proc. Natl.Acad. Sci. USA 86:5030-5034; Michiels et al. (1987) "MolecularApproaches to Genome Analysis: A Strategy for the Construction ofOrdered Overlap Clone Libraries," CABIOS 3:203-210; Olsen et al. (1986)"Random-Clone Strategy for Genomic Restriction Mapping in Yeast," Proc.Natl. Acad. Sci. USA 83:7826-7830; Craig, et al. (1990) "Ordering ofCosmid Clones Covering the Herpes Simplex Virus Type I (HSV-I) Genome: ATest Case for Fingerprinting by Hybridization," Nuc. Acids Res.18:2653-2660; and Coulson, et al. (1986) "Toward a Physical Map of theGenome of the Nematode Caenorhabditis elegans," Proc. Natl Acad. Sci.USA 83:7821-7825; each of which is hereby incorporated herein byreference.

Fingerprinting analysis also provides a means of identification. Inaddition to its value in apprehension of criminals from whom abiological sample, e.g., blood, has been collected, fingerprinting canensure personal identification for other reasons. For example, it may beuseful for identification of bodies in tragedies such as fire, flood,and vehicle crashes. In other cases the identification may be useful inidentification of persons suffering from amnesia, or of missing persons.Other forensics applications include establishing the identity of aperson, e.g., military identification "dog tags", or may be used inidentifying the source of particular biological samples. Fingerprintingtechnology is described, e.g., in Carrano, et al. (1989) "AHigh-Resolution, Fluorescence-Based, Semi-automated method for DNAFingerprinting," Genomics 4: 129-136, which is hereby incorporatedherein by reference. See, e.g., table I, for nucleic acid applications,and corresponding applications may be accomplished using polypeptides.

TABLE I VLSIPS™ TECHNOLOGY IN NUCLEIC ACIDS

I. Construction of Chips

II. Applications

A. Sequencing

1. Primary sequencing

2. Secondary sequencing (sequence checking)

3. Large scale mapping

4. Fingerprinting

B. Duplex/Triplex formation

1. Antisense

2. Sequence specific function modulation

(e.g. promoter inhibition)

C. Diagnosis

1. Genetic markers

2. Type markers

a. Blood donors

b. Tissue transplants

D. Microbiology

1. Clinical microbiology

2. Food microbiology

III. Instrumentation

A. Chip machines

B. Detection

IV. Software Development

A. Instrumentation software

B. Data reduction software

C. Sequence analysis software

The fingerprinting analysis may be used to perform various types ofgenetic screening. For example, a single substrate may be generated witha plurality of screening probes, allowing for the simultaneous geneticscreening for a large number of genetic markers. Thus, prenatal ordiagnostic screening can be simplified, economized, and made moregenerally accessible.

In addition to the sequencing, fingerprinting, and mapping applications,the present invention also provides means for determining specificity ofinteraction with particular sequences. Many of these applications weredescribed in Ser. No. 07/362,901, now abandoned, Pirrung et al. (1992)U.S. Pat. No. 5,143,854; Ser. No. 07/435,316, and Ser. No. 07/612,671.

E. Detection Methods and Apparatus

An appropriate detection method applicable to the selected labelingmethod can be selected. Suitable labels include radionucleotides,enzymes, substrates, cofactors, inhibitors, magnetic particles, heavymetal atoms, and particularly fluorescers, chemiluminescers, andspectroscopic labels. Patents teaching the use of such labels includeU.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;4,275,149; and 4,366,241.

With an appropriate label selected, the detection system best adaptedfor high resolution and high sensitivity detection may be selected. Asindicated above, an optically detectable system, e.g., fluorescence orchemiluminescence would be preferred. Other detection systems may beadapted to the purpose, e.g., electron microscopy, scanning electronmicroscopy (SEM), scanning tunneling electron microscopy (STEM),infrared microscopy, atomic force microscopy (AFM), electricalcondutance, and image plate transfer.

With a detection method selected, an apparatus for scanning thesubstrate will be designed. Apparatus, as described in Ser. No.07/362,901, now abandoned; or Pirrung et al. (1992) U.S. Pat. No.5,143,854; or Ser. No. 07/624,120, now abandoned, are particularlyappropriate. Design modifications may also be incorporated therein.

F. Data Analysis

Data is analyzed by processes similar to those described below in thesection describing theoretical analysis. More efficient algorithms willbe mathematically devised, and will usually be designed to be performedon a computer. Various computer programs which may more quickly orefficiently make measurement samples and distinguish signal from noisewill also be devised. See, particularly, Ser. No. 07/624,120, nowabandoned.

The initial data resulting from the detection system is an array of dataindicative of fluorescent intensity versus location on the substrate.The data are typically taken over regions substantially smaller than thearea in which synthesis of a given polymer has taken place. Merely byway of example, if polymers were synthesized in squares on the substratehaving dimensions of 500 microns by 500 microns, the data may be takenover regions having dimensions of 5 microns by 5 microns. In mostpreferred embodiments, the regions over which florescence data are takenacross the substrate are less than about 1/2 the area of the regions inwhich individual polymers are synthesized, preferably less than 1/10 thearea in which a single polymer is synthesized, and most preferably lessthan 1/100 the area in which a single polymer is synthesized. Hence,within any area in which a given polymer has been synthesized, a largenumber of fluorescence data points are collected.

A plot of number of pixels versus intensity for a scan should bear arough resemblance to a bell curve, but spurious data are observed,particularly at higher intensities. Since it is desirable to use anaverage of fluorescent intensity over a given synthesis region indetermining relative binding affinity, these spurious data will tend toundesirably skew the data.

Accordingly, in one embodiment of the invention the data are correctedfor removal of these spurious data points, and an average of the datapoints is thereafter utilized in determining relative bindingefficiency. In general the data are fitted to a base curve andstatistical measures are used to remove spurious data.

In an additional analytical tool, various degeneracy reducing analoguesmay be incorporated in the hybridization probes. Various aspects of thisstrategy are described, e.g., in Macevicz, S. (1990) PCT publicationnumber WO 90/04652, which is hereby incorporated herein by reference.

II. THEORETICAL ANALYSIS

The principle of the hybridization sequencing procedure is based, inpart, upon the ability to determine overlaps of short segments. TheVLSIPS technology provides the ability to generate reagents which willsaturate the possible short subsequence recognition possibilities. Theprinciple is most easily illustrated by using a binary sequence, such asa sequence of zeros and ones. Once having illustrated the application toa binary alphabet, the principle may easily be understood to encompassthree letter, four letter, five or more letter, even 20 letteralphabets. A theoretical treatment of analysis of subsequenceinformation to reconstruction of a target sequence is provided, e.e., inLysov, Yu., et al. (1988) Doklady Akademi. Nauk. SSR 303:1508-1511;Khrapko K., et al. (1989) FEBS Letters 256:118-122; Pevzner, P. (1989)J. of Biomolecular Structure and Dynamics 7:63-69; and Drmanac, R. etal. (1989) Genomics 4:114-128; each of which is hereby incorporatedherein by reference.

The reagents for recognizing the subsequences will usually be specificfor recognizing a particular polymer subsequence anywhere within atarget polymer. It is preferable that conditions may be devised whichallow absolute discrimination between high fidelity matching and verylow levels of mismatching. The reagent interaction will preferablyexhibit no sensitivity to flanking sequences, to the subsequenceposition within the target, or to any other remote structure within thesequence. For polynucleotide sequencing, the specific reagents can beoligonucleotide probes; for polypeptides and carbohydrates, antibodieswill be useful reagents. Antibody reagents should also be useful forother types of polymers.

A. Simple n-mer Structure: Theory

1. Simple two letter alphabet: example

A simple example is presented below of how a sequence of ten digitscomprising zeros and ones would be sequenceable using short segments offive digits. For example, consider the sample ten digit sequence:

1010011100.

A VLSIPS™ Technology substrate could be constructed, as discussedelsewhere, which would have reagents attached in a defined matrixpattern which specifically recognize each of the possible five digitsequences of ones and zeros. The number of possible five digitsubsequences is 2⁵ =32. The number of possible different sequences 10digits long is 2¹⁰ =1,024. The five contiguous digit subsequences withina ten digit sequence number six, i.e., positioned at digits 1-5, 2-6,3-7, 4-8, 5-9, and 6-10. It will be noted that the specific order of thedigits in the sequence is important and that the order is directional,e.g., running left to right versus right to left.

The first five digit sequence contained in the target sequence is 10100.The second is 01001, the third is 10011, the fourth is 00111, the fifthis 01110, and the sixth is 11100.

The VLSIPS™ substrate would have a matrix pattern of positionallyattached reagents which recognize each of the different 5-mersubsequences. Those reagents which recognize each of the 6 contained5-mers will bind the target, and a label allows the positionaldetermination of where the sequence specific interaction has occurred.By correlation of the position in the matrix pattern, the correspondingbound subsequences can be determined.

In the above-mentioned sequence, six different 5-mer sequences would bedetermined to be present. They would be:

    ______________________________________    10100    01001    10011     00111     01110      11100    ______________________________________

Any sequence which contains the first five digit sequence, 10100,already narrows the number of possible sequences (e.g., from 1024possible sequences) which contain it to less than about 192 possiblesequences.

This 192 is derived from the observation that with the subsequence 10100at the far left of the sequence, in positions 1-5, there are only 32possible sequences. Likewise, for that particular subsequence inpositions 2-6, 3-7, 4-8, 5-9, and 6-10. So, to sum up all of thesequences that could contain 10100, there are 32 for each position and 6positions for a total of about 192 possible sequences. However, some ofthese 10 digit sequences will have been counted twice. Thus, by virtueof containing the 10100 subsequence, the number of possible 10-mersequences has been decreased from 1024 sequences to less than about 192sequences.

In this example, not only do we know that the sequence contains 10100,but we also know that it contains the second five character sequence,01001. By virtue of knowing that the sequence contains 10100, we canlook specifically to determine whether the sequence contains asubsequence of five characters which contains the four leftmost digitsplus a next digit to the left. For example, we would look for a sequenceof X1010, but we find that there is none. Thus, we know that the 10100must be at the left end of the 10-mer. We would also look to see whetherthe sequence contains the rightmost four digits plus a next digit to theright, e.g., 0100X. We find that the sequence also contains the sequence01001, and that X is a 1. Thus, we know at least that our targetsequence has an overlap of 0100 and has the left terminal sequence101001.

Applying the same procedure to the second 5-mer, we also know that thesequence must include a sequence of five digits having the sequence1001Y where Y must be either 0 or 1. We look through the fragments andwe see that we have a 10011 sequence within our target, thus Y isalso 1. Thus, we would know that our sequence has a sequence of thefirst seven being 1010011.

Moving to the next 5-mer, we know that there must be a sequence of0011Z, where Z must be either 0 or 1. We look at the fragments producedabove and see that the target sequence contains a 00111 subsequence andZ is 1. Thus, we know the sequence must start with 10100111.

The next 5-mer must be of the sequence 0111W where W must be 0 or 1.Again, looking up at the fragments produced, we see that the targetsequence contains a 01110 subsequence, and W is a 0. Thus, our sequenceto this point is 101001110. We know that the last 5-mer must be either11100 or 11101. Looking above, we see that it is 11100 and that must bethe last of our sequence. Thus, we have determined that our sequencemust have been 1010011100.

However, it will be recognized from the example above with the sequencesprovided therein, that the sequence analysis can start with any knownpositive probe subsequence. The determination may be performed by movinglinearly along the sequence checking the known sequence with a limitednumber of next positions. Given this possibility, the sequence may bedetermined, besides by scanning all possible oligonucleotide probepositions, by specifically looking only where the next possiblepositions would be. This may increase the complexity of the scanning butmay provide a longer time span dedicated towards scanning and detectingspecific positions of interest relative to other sequence possibilities.Thus, the scanning apparatus could be set up to work its way along asequence from a given contained oligonucleotide to only look at thosepositions on the substrate which are expected to have a positive signal.

It is seen that given a sequence, it can be deconstructed into n-mers toproduce a set of internal contiguous subsequences. From any given targetsequence, we would be able to determine what fragments would result. Thehybridization sequence method depends, in part, upon being able to workin the reverse, from a set of fragments of known sequences to the fullsequence. In simple cases, one is able to start at a single position andwork in either or both directions towards the ends of the sequence asillustrated in the example.

The number of possible sequences of a given length increases veryquickly with the length of that sequence. Thus, a 10-mer of zeros andones has 1024 possibilities, a 12-mer has 4096. A 20-mer has over amillion possibilities, and a 30-mer has over a billion. However, a given30-mer has, at most, 26 different internal 5-mer sequences. Thus, a 30character target sequence having over a million possible sequences canbe substantially defined by only 26 different 5-mers. It will berecognized that the probe oligonucleotides will preferably, but need notnecessarily, be of identical length, and that the probe sequences neednot necessarily be contiguous in that the overlapping subsequences neednot differ by only a single subunit. Moreover, each position of thematrix pattern need not be homogeneous, but may actually contain aplurality of probes of known sequence. In addition, although all of thepossible subsequence specifications would be preferred, a less than fullset of sequences specifications could be used. In particular, although asubstantial fraction will preferably be at least about 70%, it may beless than that. About 20% would be preferred, more preferably at leastabout 30% would be desired. Higher percentages would be especiallypreferred.

2. Example of four letter alphabet

A four letter alphabet may be conceptualized in at least two differentways from the two letter alphabet. One way is to consider the fourpossible values at each position and to analogize in a similar fashionto the binary example each of the overlaps. A second way is to group thebinary digits into groups.

Using the first means, the overlap comparisons are performed with a fourletter alphabet rather than a two letter alphabet. Then, in contrast tothe binary system with 10 positions where 2¹⁰ =1024 possible sequences,in a 4-character alphabet with 10 positions, there will actually be 4¹⁰=1,048,576 possible sequences. Thus, the complexity of a four charactersequence has a much larger number of possible sequences compared to atwo character sequence. Note, however, that there are still only 6different internal 5-mers. For simplicity, we shall examine a 5character string with 3 character subsequences. Instead of only 1 and 0,the characters may be designated, e.g., A, C, G, and T. Let us take thesequence GGCTA. The 3-mer subsequences are:

    ______________________________________    10100    01001    10011     00111     01110      11100    ______________________________________

Given these subsequences, there is one sequence, or at most only a fewsequences which would produce that combination of subsequences, i.e.,GGCTA.

Alternatively, with a four character universe, the binary system can belooked at in pairs of digits. The pairs would be 00, 01, 10, and 11. Inthis manner, the earlier used sequence 1010011100 is looked at as10,10,01,11,00. Then the first character of two digits is selected fromthe possible universe of the four representations 00, 01, 10, and 11.Then a probe would be in an even number of digits, e.g., not fivedigits, but, three pairs of digits or six digits. A similar comparisonis performed and the possible overlaps determined. The 3-pairsubsequences are:

    ______________________________________    10100    01001    10011     00111     01110      11100    ______________________________________

and the overlap reconstruction produces 10,10,01,11,00.

The latter of the two conceptual views of the 4 letter alphabet providesa representation which is similar to what would be provided in a digitalcomputer. The applicability to a four nucleotide alphabet is easily seenby assigning, e.g., 00 to A, 01 to C, 10 to G, and 11 to T. And, infact, if such a correspondence is used, both examples for the 4character sequences can be seen to represent the same target sequence.The applicability of the hybridization method and its analysis fordetermining the ultimate sequence is easily seen if A is therepresentation of adenine, C is the representation of cytosine, G is therepresentation of guanine, and T is the representation of thymine oruracil.

3. Generalization to m-letter Alphabet

This reconstruction process may be applied to polymers of virtually anynumber of possible characters in the alphabet, and for virtually anylength sequence to be sequenced, though limitations, as discussed below,will limit its efficiency at various extremes of length. It will berecognized that the theory can be applied to a large diversity ofsystems where sequence is important.

For example, the method could be applied to sequencing of a polypeptide.A polypeptide can have any of twenty natural amino acid possibilities ateach position. A twenty letter alphabet is amenable to sequencing bythis method so long as reagents exist for recognizing shortersubsequences therein. A preferred reagent for achieving that goal wouldbe a set of monoclonal antibodies each of which recognizes a specificthree contiguous amino acid subsequence. A complete set of antibodieswhich recognize all possible subsequences of a given length, e.g., 3amino acids, and preferably with a uniform affinity, would be 20³ =8000reagents.

It will also be recognized that each target: sequence which isrecognized by the specific reagents need not have homogeneous termini.Thus, fragments of the entire target sequence will also be useful forhybridizing appropriate subsequences. It is, however, preferable thatthere not be a significant amount of labeled homogeneous contaminatingextraneous sequences. This constraint does usually require thepurification of the target molecule to be sequenced, but a specificlabel technique would dispense with a purification requirement if theunlabeled extraneous sequences do not interfere with the labeledsequences.

In addition, conformational effects of target polypeptide folding may,in certain embodiments, be negligible if the polypeptide is fragmentedinto sufficiently small peptides, or if the interaction is performedunder conditions where conformation, but not specific interaction, isdisrupted.

B. Complications

Two obvious complications exist with the method of sequence analysis byhybridization. The first results from a probe of inappropriate lengthwhile the second relates to internally repeated sequences.

The first obvious complication is a problem which arises from aninappropriate length of recognition sequence, which causes problems withthe specificity of recognition. For example, if the recognized sequenceis too short, every sequence which is utilized will be recognized byevery probe sequence. This occurs, e.g., in a binary system where theprobes are each of sequences which occur relatively frequently, e.g., atwo character probe for the binary system. Each possible two characterprobe would be expected to appear 1/4 of the time in every single twocharacter position. Thus, the above sequence example would be recognizedby each of the 00, 10, 01, and 11. Thus, the sequence information isvirtually lost because the resolution is too low and each recognitionreagent specifically binds at multiple sites on the target sequence.

The number of different probes which bind to a target depends on therelationship between the probe length-and the target length. At theextreme of short probe length, the just mentioned problem exists ofexcessive redundancy and lack of resolution. The lack of stability inrecognition will also be a problem with extremely short probes. At theextreme of long probe length, each entire probe sequence is on adifferent position of a substrate. However, a problem arises from thenumber of possible sequences, which goes up dramatically with the lengthof the sequence. Also, the specificity of recognition begins to decreaseas the contribution to binding by any particular subunit may becomesufficiently low that the system fails to distinguish the fidelity ofrecognition. Mismatched hybridization may be a problem with thepolynucleotide sequencing applications, though the fingerprinting andmapping applications may not be so strict in their fidelityrequirements. As indicated above, a thirty position binary sequence hasover a million possible sequences, a number which starts to becomeunreasonably large in its required number of different sequences, eventhough the target length is still very short. Preparing a substrate withall sequence possibilities for a long target may be extremely difficultdue to the many different oligomers which must be synthesized.

The above example illustrates how a long target sequence may bereconstructed with a reasonably small number of shorter subsequences.Since the present day resolution of the regions of the substrate havingdefined oligomer probes attached to the substrate approaches about 10microns by 10 microns for resolvable regions, about 10⁶, or 1 million,positions can be placed on a one centimeter square substrate. However,high resolution systems may have particular disadvantages which may beoutweighed using the lower density substrate matrix pattern. For thisreason, a sufficiently large number of probe sequences can be utilizedso that any given target sequence may be determined by hybridization toa relatively small number of probes.

A second complication relates to convergence of sequences to a singlesubsequence. This will occur when a particular subsequence is repeatedin the target sequence. This problem can be addressed in at least twodifferent ways. The first, and simpler way, is to separate the repeatsequences onto two different targets. Thus, each single target will nothave the repeated sequence and can be analyzed to its end. Thissolution, however, complicates the analysis by requiring that some meansfor cutting at a site between the repeats can be located. Typically acareful sequencer would want to have two intermediate cut points so thatthe intermediate region can also be sequenced in both directions acrosseach of the cut points. This problem is inherent in the hybridizationmethod for sequencing but can be minimized by using a longer known probesequence so that the frequency of probe repeats is decreased.

Knowing the sequence of flanking sequences of the repeat will simplifythe use of polymerase chain reaction (PCR) or a similar technique tofurther definitively determine the sequence between sequence repeats.Probes can be made to hybridize to those known sequences adjacent therepeat sequences, thereby producing new target sequences for analysis.See, e.g., Innis et al. (eds.) (1990) PCR Protocols: A Guide to Methodsand Applications, Academic Press; and methods for synthesis ofoligonucleotide probes, see, e.g., Gait (1984) OligonucleotideSynthesis: A Practical Approach, IRL Press, Oxford.

Other means for dealing with convergence problems include usingparticular longer probes, and using degeneracy reducing analogues, see,e.g., Macevicz, S. (1990) PCT publication number WO 90/04652, which ishereby incorporated herein by reference. By use of stretches of thedegeneracy reducing analogues with other probes in particularcombinations, the number of probes necessary to fully saturate thepossible oligomer probes is decreased. For example, with a stretch of12-mers having the central 4-mer of degenerate nucleotides, incombination with all of the possible 8-mers, the collection numberstwice the number of possible 8-mers, e.g. 65,536+65,536=131,072, but thepopulation provides screening equivalent to all possible 12-mers.

By way of further explanation, all possible oligonucleotide 8-mers maybe depicted in the fashion:

N1-N2-N3-N4-N5-N6-N7-N8,

in which there are 4⁸ =65,536 possible 8-mers. As described in Ser. No.07/624,120, now abandoned, producing all possible 8-mers requires 4×8=32chemical binary synthesis steps to produce the entire matrix pattern of65,536 8-mer possibilities. By incorporating degeneracy reducingnucleotides, D's, which hybridize nonselectively to any correspondingcomplementary nucleotide, new oligonucleotides 12-mers can be made inthe fashion:

N1-N2-N3-N4-D-D-D-D-N5-N6-N7-N8,

in which there are again, as above, only 4⁸ =65,536 possible "12-mers",which in reality only have 8 different nucleotides.

However, it can be seen that each possible 12-mer probe could berepresented by a group of the two 8-mer types. Moreover, repeats of lessthan 12 nucleotides would not converge, or cause repeat problems in theanalysis. Thus, instead of requiring a collection of probescorresponding to all 12-mers, or 4¹² =16,777,216 different 12-mers, thesame information can be derived by making 2 sets of "8-mers" consistingof the typical 8-mer collection of 4⁸ =65,536 and the "12-mer" set withthe degeneracy reducing analogues, also requiring making 4⁸ =65,536. Thecombination of the two sets, requires making 65,536+65,536=131,072different molecules, but giving the information of 16,777,216 molecules.Thus, incorporating the degeneracy reducing analogue decreases thenumber of molecules necessary to get 12-mer resolution by a factor ofabout 128-fold.

C. Non-polynucleotide Embodiments

The above example is directed towards a polynucleotide embodiment. Thisapplication is relatively easily achieved because the specific reagentswill typically be complementary oligonucleotides, although in certainembodiments other specific reagents may be desired. For example, theremay be circumstances where other than complementary base pairing will beutilized. The polynucleotide targets, will usually be single strand, butmay be double or triple stranded in various applications. However, atriple stranded specific interaction might be sometimes desired, or aprotein or other specific binding molecule may be utilized. For example,various promoter or DNA sequence specific binding proteins might beused, including, e.g., restriction enzyme binding domains, other bindingdomains, and antibodies. Thus, specific recognition reagents besidesoligonucleotides may be utilized.

For other polymer targets, the specific reagents will often bepolypeptides. These polypeptides may be protein binding domains fromenzymes or other proteins which display specificity for binding. Usuallyan antibody molecule may be used, and monoclonal antibodies may beparticularly desired. Classical methods may be applied for preparingantibodies, see, e.g., Harlow and Lane (1988) Antibodies: A LaboratoryManual Cold Spring Harbor Press, New York; and Goding (1986) MonoclonalAntibodies: Principles and Practice (2d Ed.) Academic Press, San Diego.Other suitable techniques for in vitro exposure of lymphocytes to theantigens or selection of libraries of antibody binding sites aredescribed, e.g., in Huse et al. (1989) Science 246:1275-1281; and Wardet al. 91989) Nature 341:544-546, each of which is hereby incorporatedherein by reference. Unusual antibody production methods are alsodescribed, e.g., in Hendricks et al. (1989) BioTechnology 7:1271-1274;and Hiatt et al. (1989) Nature 342:76-78, each of which is herebyincorporated herein by reference. Other molecules which may exhibitspecific binding interaction may be useful for attachment to a VLSIPSsubstrate by various methods, including the caged biotin methods, see,e.g., Ser. No. 07/435,316, now abandoned, and Barrett et al. (1993) U.S.Pat. No. 5,252,743.

The antibody specific reagents should be particularly useful for thepolypeptide, carbohydrate, and synthetic polymer applications.Individual specific reagents might be generated by an automated processto generate the number of reagents necessary to advantageously use thehigh density positional matrix pattern. In an alternative approach, aplurality of hybridoma cells may be screened for their ability to bindto a VLSIPS matrix possessing the desired sequences whose bindingspecificity is desired. Each cell might be individually grown up and itsbinding specificity determined by VLSIPS apparatus and technology. Analternative strategy would be to expose the same VLSIPS matrix to apolyclonal serum of high titer. By a successively large volume of serumand different animals, each region of the VLSIPS substrate would haveattached to it a substantial number of antibody molecules withspecificity of binding. The substrate, with non-covalently boundantibodies could be derivatized and the antibodies transferred to anadjacent second substrate in the matrix pattern in which the antibodymolecules had attached to the first matrix. If the sensitivity ofdetection of binding interaction is sufficiently high, such a lowefficiency transfer of antibody molecules may produce a sufficientlyhigh signal to be useful for many purposes, including the sequencingapplications.

In another embodiment, capillary forces may be used to transfer theselected reagents to a new matrix, to which the reagents would bepositionally attached in the pattern of the recognized sequences. Or,the reagents could be transversely electrophoresed, magneticallytransferred, or otherwise transported to a new substrate in theirretained positional pattern.

III. POLYNUCLEOTIDE SEQUENCING

In principle, the making of a substrate having a positionally definedmatrix pattern of all possible oligonucleotides of a given lengthinvolves a conceptually simple method of synthesizing each and everydifferent possible oligonucleotide, and affixing them to a definableposition. Oligonucleotide synthesis is presently mechanized and enabledby current technology, see, e.g., Ser. No. 07/362,901, now abandoned;Pirrung et al. (1992) U.S. Pat. No. 5,143,854; and instruments suppliedby Applied Biosystems, Foster City, Calif.

A. Preparation of Substrate Matrix

The production of the collection of specific oligonucleotides used inpolynucleotide sequencing may be produced in at least two differentways. Present technology certainly allows production of ten nucleotideoligomers on a solid phase or other synthesizing system. See, e.g.,instrumentation provided by Applied Biosystems, Foster City, Calif.Although a single oligonucleotide can be relatively easily made, a largecollection of them would typically require a fairly large amount of timeand investment. For example, there are 4¹⁰ =1,048,576 possible tennucleotide oligomers. Present technology allows making each and everyone of them in a separate purified form though such might be costly andlaborious.

Once the desired repertoire of possible oligomer sequences of a givenlength have been synthesized, this collection of reagents may beindividually positionally attached to a substrate, thereby allowing abatchwise hybridization step. Present technology also would allow thepossibility of attaching each and every one of these 10-mers to aseparate specific position on a solid matrix. This attachment could beautomated in any of a number of ways, particularly through the use of acaged biotin type linking. This would produce a matrix having each ofdifferent possible 10-mers.

A batchwise hybridization is much preferred because of itsreproducibility and simplicity. An automated process of attachingvarious reagents to positionally defined sites on a substrate isprovided in Pirrung et al. (1992) U.S. Pat. No. 5,143,854; Ser. No.07/624,120, now abandoned; and Barrett et al. (1993) U.S. Pat. No.5,252,743; each of which is hereby incorporated herein by reference.

Instead of separate synthesis of each oligonucleotide, theseoligonucleotides are conveniently synthesized in parallel by sequentialsynthetic processes on a defined matrix pattern as provided in Pirrunget al. (1992) U.S. Pat. No. 5,143,854; and Ser. No. 07/624,120, nowabandoned, which are incorporated herein by reference Here, theoligonucleotides are synthesized stepwise on a substrate at positionallyseparate and defined positions. Use of photosensitive blocking reagentsallows for defined sequences of synthetic steps over the surface of amatrix pattern. By use of the binary masking strategy, the surface ofthe substrate can be positioned to generate a desired pattern ofregions, each having a defined sequence oligonucleotide synthesized andimmobilized thereto.

Although the prior art technology can be used to generate the desiredrepertoire of oligonucleotide probes, an efficient and cost effectivemeans would be to use the VLSIPS technology described in Pirrung et al.(1992) U.S. Pat. No. 5,143,854 and Ser. No. 07/624,120, now abandoned.In this embodiment, the photosensitive reagents involved in theproduction of such a matrix are described below.

The regions for synthesis may be very small, usually less than about 100μm×100 μm, more usually less than about 50 μm×50 μm. Thephotolithography technology allows synthetic regions of less than about10 μm×10 μm, about 3 μm×3 μm, or less. The detection also may detectsuch sized regions, though larger areas are more easily and reliablymeasured.

At a size of about 30 microns by 30 microns, one million regions wouldtake about 11 centimeters square or a single wafer of about 4centimeters by 4 centimeters. Thus the present technology provides formaking a single matrix of that size having all one million plus possibleoligonucleotides. Region size is sufficiently small to correspond todensities of at least about 5 regions/cm², 20 regions/cm², 50regions/cm², 100 regions/cm², and greater, including 300 regions/cm²,1000 regions/cm², 3K regions/cm², 10K regions/cm², 30K regions/cm², 100Kregions/cm², 300K regions/cm² or more, even in excess of one millionregions/cm².

Although the pattern of the regions which contain specific sequences istheoretically not important, for practical reasons certain patterns willbe preferred in synthesizing the oligonucleotides. The application ofbinary masking algorithms for generating the pattern of knownoligonucleotide probes is described in related Ser. No. 07/624,120, nowabandoned, which was filed simultaneously with this application. By useof these binary masks, a highly efficient means is provided forproducing the substrate with the desired matrix pattern of differentsequences. Although the binary masking strategy allows for the synthesisof all lengths of polymers, the strategy may be easily modified toprovide only polymers of a given length. This is achieved by omittingsteps where a subunit is not attached.

The strategy for generating a specific pattern may take any of a numberof different approaches. These approaches are well described in relatedapplication Ser. No. 07/624,120, now abandoned, and include a number ofbinary masking approaches which will not be exhaustively discussedherein. However, the binary masking and binary synthesis approachesprovide a maximum of diversity with a minimum number of actual syntheticsteps.

The length of oligonucleotides used in sequencing applications will beselected on criteria determined to some extent by the practical limitsdiscussed above. For example, if probes are made as oligonucleotides,there will be 65,536 possible eight nucleotide sequences. If a ninesubunit oligonucleotide is selected, there are 262,144 possiblepermeations of sequences. If a ten-mer oligonucleotide is selected,there are 1,048,576 possible permeations of sequences. As the numbergets larger, the required number of positionally defined subunitsnecessary to saturate the possibilities also increases. With respect tohybridization conditions, the length of the matching necessary to conferstability of the conditions selected can be compensated for. See, e.g.,Kanehisa, M. (1984) Nuc. Acids Res. 12:203-213, which is herebyincorporated herein by reference.

Although not described in detail here, but below for oligonucleotideprobes, the VLSIPS technology would typically use a photosensitiveprotective group on an oligonucleotide. Sample oligonucleotides areshown in FIG. 1. In particular, the photoprotective group on thenucleotide molecules may be selected from a wide variety of positivelight reactive groups preferably including nitro aromatic compounds suchas o-nitro-benzyl derivatives or benzylsulfonyl. See, e.g., Gait (1984)Oligonucleotide Synthesis: A Practical Approach, IRL Press, Oxford,which is hereby incorporated herein by reference. In a preferredembodiment, 6-nitro-veratryl oxycarbony (NVOC), 2-nitrobenzyloxycarbonyl (NBOC), or α,α-dimethyl-dimethoxybenzyl oxycarbonyl (DEZ) isused. Photoremovable protective groups are described in, e.g.,Patchornik (1970) J. Amer. Chem. Soc. 92:6333-6335; and Amit et al.(1974) J. Organic Chem. 39:192-196; each of which is hereby incorporatedherein by reference.

A preferred linker for attaching the oligonucleotide to a silicon matrixis illustrated in FIG. 2. A more detailed description is provided below.A photosensitive blocked nucleotide may be attached to specificlocations of unblocked prior cycles of attachments on the substrate andcan be successively built up to the correct length oligonucleotideprobe.

It should be noted that multiple substrates may be simultaneouslyexposed to a single target sequence where each substrate is a duplicateof one another or where, in combination, multiple substrates togetherprovide the complete or desired subset of possible subsequences. Thisprovides the opportunity to overcome a limitation of the density ofpositions on a single substrate by using multiple substrates. In theextreme case, each probe might be attached to a single bead or substrateand the beads sorted by whether there is a binding interaction. Thosebeads which do bind might be encoded to indicate the subsequencespecificity of reagents attached thereto.

Then, the target may be bound to the whole collection of beads and thosebeads that have appropriate specific reagents on them will bind to thetarget. Then a sorting system may be utilized to sort those beads thatactually bind the target from those that do not. This may beaccomplished by presently available cell sorting devices or a similarapparatus. After the relatively small number of beads which have boundthe target have been collected, the encoding scheme may be read off todetermine the specificity of the reagent on the bead. An encoding systemmay include a magnetic system, a shape encoding system, a color encodingsystem, or a combination of any of these, or any other encoding system.Once again, with the collection of specific interactions that haveoccurred, the binding may be analyzed for sequence information,fingerprint information, or mapping information.

The parameters of polynucleotide sizes of both the probes and targetsequences are determined by the applications and other circumstances.The length of the oligonucleotide probes used will depend in part uponthe limitations of the VLSIPS technology to provide the number ofdesired probes. For example, in an absolute sequencing application, itis often useful to have virtually all of the possible oligonucleotidesof a given length. As indicated above, there are 65,536 8-mers, 262,1449-mers, 1,048,576 10-mers, 4,194,304 11-mers, etc. As the length of theoligomer increases the number of different probes which must besynthesized also increases at a rate of a factor of 4 for everyadditional nucleotide. Eventually the size of the matrix and thelimitations in the resolution of regions in the matrix will reach thepoint where an increase in number of probes becomes disadvantageous.However, this sequencing procedure requires that the system be able todistinguish, by appropriate selection of hybridization and washingconditions, between binding of absolute fidelity and binding ofcomplementary sequences containing mismatches. On the other hand, if thefidelity is unnecessary, this discrimination is also unnecessary and asignificantly longer probe may be used. Significantly longer probeswould typically be useful in fingerprinting or mapping applications.

The length of the probe is selected for a length that will allow theprobe to bind with specificity to possible targets. The hybridizationconditions are also very important in that they will determine howclosely the homology of complementary binding will be detected. In fact,a single target may be evaluated at a number of different conditions todetermine its spectrum of specificity for binding particular probes.This may find use in a number of other applications besides thepolynucleotide sequencing fingerprinting or mapping. For example, itwill be desired to determine the spectrum of binding affinities andspecificities of cell surface antigens with binding by particularantibodies immobilized on the substrate surface, particularly underdifferent interaction conditions. In a related fashion, differentregions with reagents having differing affinities or levels ofspecificity may allow such a spectrum to be defined using a singleincubation, where various regions, at a given hybridization condition,show the binding affinity. For example, fingerprint probes of variouslengths, or with specific defined non-matches may be used. Unnaturalnucleotides or nucleotides exhibiting modified specificity ofcomplementary binding are described in greater detail in Macevicz (1990)PCT pub. No. WO 90/04652; and see the section on modified nucleotides inthe Sigma Chemical Company catalogue.

B. Labeling Target Nucleotide

The label used to detect the target sequences will be determined, inpart, by the detection methods being applied. Thus, the labeling methodand label used are selected in combination with the actual detectingsystems being used.

Once a particular label has been selected, appropriate labelingprotocols will be applied, as described below for specific embodiments.Standard labeling protocols for nucleic acids are described, e.g., inSambrook et al.; Kambara, H. et al. (1988) BioTechnology 6:816-821;Smith, L. et al. (1985) Nuc. Acids Res. 13:2399-2412; for polypeptides,see, e.g., Allen G. (1989) Sequencing of Proteins and Peptides,Elsevier, New York, especially chapter 5, and Greenstein and Winitz(1961) Chemistry of the Amino Acids, Wiley and Sons, New York.Carbohydrate labeling is described, e.g., in Chaplin and Kennedy (1986)Carbohydrate Analysis: A Practical Approach, IRL Press, Oxford. Labelingof other polymers will be performed by methods applicable to them asrecognized by a person having ordinary skill in manipulating thecorresponding polymer.

In some embodiments, the target need not actually be labeled if a meansfor detecting where interaction takes place is available. As describedbelow, for a nucleic acid embodiment, such may be provided by anintercalating dye which intercalates only into double stranded segments,e.g., where interaction occurs. See, e.g., Sheldon et al. U.S. Pat. No.4,582,789.

In many uses, the target sequence will be absolutely homogeneous, bothwith respect to the total sequence and with respect to the ends of eachmolecule. Homogeneity with respect to sequence is important to avoidambiguity. It is preferable that the target sequences of interest not becontaminated with a significant amount of labeled contaminatingsequences. The extent of allowable contamination will depend on thesensitivity of the detection system and the inherent signal to noise ofthe system. Homogeneous contamination sequences will be particularlydisruptive of the sequencing procedure.

However, although the target polynucleotide must have a unique sequence,the target molecules need not have identical ends. In fact, thehomogeneous target molecule preparation may be randomly sheared toincrease the numerical number of molecules. Since the total informationcontent remains the same, the shearing results only in a higher numberof distinct sequences which may be labeled and bind to the probe. Thisfragmentation may give a vastly superior signal relative to apreparation of the target molecules having homogeneous ends. The signalfor the hybridization is likely to be dependent on the numericalfrequency of the target-probe interactions. If a sequence isindividually found on a larger number of separate molecules a bettersignal will result. In fact, shearing a homogeneous preparation of thetarget may often be preferred before the labeling procedure isperformed, thereby producing a large number of labeling groupsassociated with each subsequence.

C. Hybridization Conditions

The hybridization conditions between probe and target should be selectedsuch that the specific recognition interaction, i.e., hybridization, ofthe two molecules is both sufficiently specific and sufficiently stable.See, e.g., Hames and Higgins (1985) Nucleic Acid Hybridisation: APractical Approach, IRL Press, Oxford. These conditions will bedependent both on the specific sequence and often on the guanine andcytosine (GC) content of the complementary hybrid strands. Theconditions may often be selected to be universally equally stableindependent of the specific sequences involved. This typically will makeuse of a reagent such as an alkylammonium buffer. See, Wood et al.(1985) "Base Composition-independent Hybridization inTetramethylammonium Chloride: A Method for Oligonucleotide Screening ofHighly Complex Gene Libraries," Proc. Natl. Acad. Sci. USA,82:1585-1588; and Krupov et al. (1989) "An Oligonucleotide HybridizationApproach to DNA Sequencing," FEBS Letters, 256:118-122; each of which ishereby incorporated herein by reference. An alkylammonium buffer tendsto minimize differences in hybridization rate and stability due to GCcontent. By virtue of the fact that sequences then hybridize withapproximately equal affinity and stability, there is relatively littlebias in strength or kinetics of binding for particular sequences.Temperature and salt conditions along with other buffer parametersshould be selected such that the kinetics of renaturation should beessentially independent of the specific target subsequence oroligonucleotide probe involved. In order to ensure this, thehybridization reactions will usually be performed in a single incubationof all the substrate matrices together exposed to the identical sametarget probe solution under the same conditions.

Alternatively, various substrates may be individually treateddifferently. Different substrates may be produced, each having reagentswhich bind to target subsequences with substantially identicalstabilities and kinetics of hybridization. For example, all of the highGC content probes could be synthesized on a single substrate which istreated accordingly. In this embodiment, the arylammonium buffers couldbe unnecessary. Each substrate is then treated in a manner such that thecollection of substrates show essentially uniform binding and thehybridization data of target binding to the individual substrate matrixis combined with the data from other substrates to derive the necessarysubsequence binding information. The hybridization conditions willusually be selected to be sufficiently specific such that the fidelityof base matching will be properly discriminated. Of course, controlhybridizations should be included to determine the stringency andkinetics of hybridization.

D. Detection; VLSIPS™ Technology Scanning

The next step of the sequencing process by hybridization involveslabeling of target polynucleotide molecules. A quickly and easilydetectable signal is preferred. The VLSIPS™ Technology apparatus isdesigned to easily detect a fluorescent label, so fluorescent tagging ofthe target sequence is preferred. Other suitable labels include heavymetal labels, magnetic probes, chromogenic labels (e.g., phosphorescentlabels, dyes, and fluorophores) spectroscopic labels, enzyme linkedlabels, radioactive labels, and labeled binding proteins. Additionallabels are described in U.S. Pat. No. 4,366,241, which is incorporatedherein by reference.

The detection methods used to determine where hybridization has takenplace will typically depend upon the label selected above. Thus, for afluorescent label a fluorescent detection step will typically be used.Pirrung et al. (1992) U.S. Pat. No. 5,143,854 and Ser. No. 07/624,120,now abandoned, describe apparatus and mechanisms for scanning asubstrate matrix using fluorescence detection, but a similar apparatusis adaptable for other optically detectable labels.

The detection method provides a positional localization of the regionwhere hybridization has taken place. However, the position is correlatedwith the specific sequence of the probe since the probe has specificallybeen attached or synthesized at a defined substrate matrix position.Having collected all of the data indicating the subsequences present inthe target sequence, this data may be aligned by overlap to reconstructthe entire sequence of the target, as illustrated above.

It is also possible to dispense with actual labeling if some means fordetecting the positions of interaction between the sequence specificreagent and the target molecule are available. This may take the form ofan additional reagent which can indicate the sites either ofinteraction, or the sites of lack of interaction, e.g., a negativelabel. For the nucleic acid embodiments, locations of double strandinteraction may be detected by the incorporation of intercalating dyes,or other reagents such as antibody or other reagents that recognizehelix formation, see, e.g., Sheldon, et al. (1986) U.S. Pat. No.4,582,789, which is hereby incorporated herein by reference.

E. Analysis

Although the reconstruction can be performed manually as illustratedabove, a computer program will typically be used to perform the overlapanalysis. A program may be written and run on any of a large number ofdifferent computer hardware systems. The variety of operating systemsand languages useable will be recognized by a computer softwareengineer. Various different languages may be used, e.g., BASIC; C;PASCAL; etc. A simple flow chart of data analysis is illustrated in FIG.4.

F. Substrate Reuse

Finally, after a particular sequence has been hybridized and the patternof hybridization analyzed, the matrix substrate should be reusable andreadily prepared for exposure to a second or subsequent targetpolynucleotides. In order to do so, the hybrid duplexes are disruptedand the matrix treated in a way which removes all traces of the originaltarget. The matrix may be treated with various detergents or solvents towhich the substrate, the oligonucleotide probes, and the linkages to thesubstrate are inert. This treatment may include an elevated temperaturetreatment, treatment with organic or inorganic solvents, modificationsin pH, and other means for disrupting specific interaction. Thereafter,a second target may actually be applied to the recycled matrix andanalyzed as before.

G. Non-Polynucleotide Aspects

Although the sequencing, fingerprinting, and mapping functions will makeuse of the natural sequence recognition property of complementarynucleotide sequences, the non-polynucleotide sequences typically requireother sequence recognition reagents. These reagents will take the form,typically, of proteins exhibiting binding specificity, e.g., enzymebinding sites or antibody binding sites.

Enzyme binding sites may be derived from promoter proteins, restrictionenzymes, and the like. See, e.g., Stryer, L. (1988) Biochemistry, W. H.Freeman, Palo Alto. Antibodies will typically be produced using standardprocedures, see, e.g., Harlow and Lane (1988) Antibodies: A LaboratoryManual, Cold Spring Harbor Press, New York; and Goding (1986) MonoclonalAntibodies: Principles and Practice, (2d Ed.) Academic Press, San Diego.

Typically, an antigen, or collection of antigens are presented to animmune system. This may take the form of synthesized short polymersproduced by the VLSIPS technology, or by the other synthetic means, orfrom isolation of natural products. For example, antigen for thepolypeptides may be made by the VLSIPS technology, by standard peptidesynthesis, by isolation of natural proteins with or without degradationto shorter segments, or by expression of a collection of short nucleicacids of random or defined sequences. See, e.g., Tuerk and Gold (1990)Science 249:505-510, for generation of a collection of randomlymutagenized oligonucleotides useful for expression.

The antigen or collection is presented to an appropriate immune system,e.g., to a whole animal as in a standard immunization protocol, or to acollection of immune cells or equivalent. In particular, see Ward et al.(1989) Nature 341:544-546; and Huse et al. (1989) Science 246:1275-1281,each of which is hereby incorporated herein by reference.

A large diversity of antibodies will be generated, some of which havespecificities for the desired sequences. Antibodies may be purifiedhaving the desired sequence specificities by isolating the cellsproducing them. For example, a VLSIPS substrate with the desiredantigens synthesized thereon may be used to isolate cells with cellsurface reagents which recognize the antigens. The VLSIPS substrate maybe used as an affinity reagent to select and recover the appropriatecells. Antibodies from those cells may be attached to a substrate usingthe caged biotin methodology, or by attaching a targeting molecule,e.g., an oligonucleotide. Alternatively, the supernatants from antibodyproducing cells can be easily assayed using a VLSIPS substrate toidentify the cells producing the appropriate antibodies.

Although cells may be isolated, specific antibody molecules whichperform the sequence recognition will also be sufficient. Preferablypopulations of antibody with a known specificity can be isolated.Supernatants from a large population of producing cells may be passedover a VLSIPS substrate to bind to the desired antigens attached to thesubstrate. When a sufficient density of antibody molecules are attached,they may be removed by an automated process, preferably as antibodypopulations exhibiting specificity of binding.

In one particular embodiment, a VLSIPS substrate, e.g., with a largeplurality of fingerprint antigens attached thereto, is used to isolateantibodies from a supernatant of a population of cells producingantibodies to the antigens. Using the substrate as an affinity reagent,the antibodies will attach to the appropriate positionally definedantigens. The antibodies may be carefully removed therefrom, preferablyby an automated system which retains their homogeneous specificities.The isolated antibodies can be attached to a new substrate in apositionally defined matrix pattern.

In a further embodiment, these spatially separated antibodies may beisolated using a specific targeting method for isolation. In thisembodiment, a linker molecule which attaches to a particular portion ofthe antibody, preferably away from the binding site, can be attached tothe antibodies. Various reagents will be used, including staphylococcusprotein A or antibodies which bind to domains remote from the bindingsite. Alternatively, the antibodies in the population, before affinitypurification, may be derivatized with an appropriate reagent compatiblewith new VLSIPS synthesis. A preferred reagent is a nucleotide which canserve as a linker to synthetic VLSIPS steps for synthesizing a specificsequence thereon. Then, by successive VLSIPS cycles, each of theantibodies attached to the defined antigen regions can have a definedoligonucleotide synthesized thereon and corresponding in area to theregion of the substrate having each antigen attached. These definedoligonucleotides will be useful as targeting reagents to attach thoseantibodies possessing the same target sequence specificity at definedpositions on a new substrate, by virtue of having bound to the antigenregion, to a new VLSIPS substrate having the complementary targetoligonucleotides positionally located on it. In this fashion, a VLSIPSsubstrate having the desired antigens attached thereto can be used togenerate a second VLSIPS substrate with positionally defined reagentswhich recognize those antigens.

The selected antigens will typically be selected to be those whichdefine particular functionalities or properties, so as to be useful forfingerprinting and other uses. They will also be useful for mapping andsequencing embodiments.

IV. FINGERPRINTING

A. General

Many of the procedures and techniques used in the polynucleotidesequencing section are also appropriate for fingerprinting applications.See, e.g., Poustka, et al. (1986) Cold Spring Harbor Symposia on Quant.Biol., vol. LI, 131-139, Cold Spring Harbor Press, New York; which ishereby incorporated herein by reference. The fingerprinting methodprovided herein is based, in part, upon the ability to positionallylocalize a large number of different specific probes onto a singlesubstrate. This high density matrix pattern provides the ability toscreen for, or detect, a very large number of different sequencessimultaneously. In fact, depending upon the hybridization conditions,fingerprinting to the resolution of virtually absolute matching ofsequence is possible thereby approaching an absolute sequencingembodiment. And the sequencing embodiment is very useful in identifyingthe probes useful in further fingerprinting uses. For example,characteristic features of genetic sequences will be identified as beingdiagnostic of the entire sequence. However, in most embodiments, longerprobe and target will be used, and for which slight mismatching may notneed to be resolved.

B. Preparation of Substrate Matrix

A collection of specific probes may be produced by either of the methodsdescribed above in the section on sequencing. Specific oligonucleotideprobes of desired lengths may be individually synthesized on a standardoligonucleotide synthesizer. The length of these probes is limited onlyby the ability of the synthesizer to continue to accurately synthesize amolecule. Oligonucleotides or sequence fragments may also be isolatedfrom natural sources. Biological amplification methods may be coupledwith synthetic synthesizing procedures such as, e.g., polymerase chainreaction.

In one embodiment, the individually isolated probes may be attached tothe matrix at defined positions. These probe reagents may be attached byan automated process making use of the caged biotin methodologydescribed in Ser. No. 07/612,671, or using photochemical reagents, see,e.g., Dattagupta et al. (1985) U.S. Pat. No. 4,542,102 and (1987) U.S.Pat. No. 4,713,326. Each individually purified reagent can be attachedindividually at specific locations on a substrate.

In another embodiment, the VLSIPS synthesizing technique may be used tosynthesize the desired probes at specific positions on a substrate. Theprobes may be synthesized by successively adding appropriate monomersubunits, e.g., nucleotides, to generate the desired sequences.

In another embodiment, a relatively short specific oligonucleotide isused which serves as a targeting reagent for positionally directing thesequence recognition reagent. For example, the sequence specificreagents having a separate additional sequence recognition segment(usually of a different polymer from the target sequence) can bedirected to target oligonucleotides attached to the substrate. By use ofnon-natural targeting reagents, e.g., unusual nucleotide analogues whichpair with other unnatural nucleotide analogues and which do notinterfere with natural nucleotide interactions, the natural andnon-natural portions can coexist on the same molecule withoutinterfering with their individual functionalities. This can combine botha synthetic and biological production system analogous to the techniquefor targeting monoclonal antibodies to locations on a VLSIPS substrateat defined positions. Unnatural optical isomers of nucleotides may beuseful unnatural reagents subject to similar chemistry, but incapable ofinterfering with the natural biological polymers. See also, Ser. No.07/626,730, which is hereby incorporated herein by reference.

After the separate substrate attached reagents are attached to thetargeting segment, the two are crosslinked, thereby permanentlyattaching them to the substrate. Suitable crosslinking reagents areknown, see, e.g., Dattagupta et al. (1985) U.S. Pat. No. 4,542,102 and(1987) "Coupling of nucleic acids to solid support by photochemicalmethods," U.S. Pat. No. 4,713,326, each of which is hereby incorporatedherein by reference. Similar linkages for attachment of proteins to asolid substrate are provided, e.g., in Merrifield (1986) Science232:341-347, which is hereby incorporated herein by reference.

C. Labeling Target Nucleotides

The labeling procedures used in the sequencing embodiments will also beapplicable in the fingerprinting embodiments. However, since thefingerprinting embodiments often will involve relatively large targetmolecules and relatively short oligonucleotide probes, the amount ofsignal necessary to incorporate into the target sequence may be lesscritical than in the sequencing applications. For example, a relativelylong target with a relatively small number of labels per molecule may beeasily amplified or detected because of the relatively large targetmolecule size.

In various embodiments, it may be desired to cleave the target intosmaller segments as in the sequencing embodiments. The labelingprocedures and cleavage techniques described in the sequencingembodiments would usually also be applicable here.

D. Hybridization Conditions

The hybridization conditions used in fingerprinting embodiments willtypically be less critical than for the sequencing embodiments. Thereason is that the amount of mismatching which may be useful inproviding the fingerprinting information would typically be far greaterthan that necessary in sequencing uses. For example, Southernhybridizations do not typically distinguish between slightly mismatchedsequences. Under these circumstances, important and valuable informationmay be arrived at with less stringent hybridization conditions whileproviding valuable fingerprinting information. However, since the entiresubstrate is typically exposed to the target molecule at one time, thebinding affinity of the probes should usually be of approximatelycomparable levels. For this reason, if oligonucleotide probes are beingused, their lengths should be approximately comparable and will beselected to hybridize under conditions which are common for most of theprobes on the substrate. Much as in a Southern hybridization, the targetand oligonucleotide probes are of lengths typically greater than about25 nucleotides. Under appropriate hybridization conditions, e.g.,typically higher salt and lower temperature, the probes will hybridizeirrespective of imperfect complementarity. In fact, with probes ofgreater than, e.g., about fifty nucleotides, the difference in stabilityof different sized probes will be relatively minor.

Typically the fingerprinting is merely for probing similarity orhomology. Thus, the stringency of hybridization can usually be decreasedto fairly low levels. See, e.g., Wetmur and Davidson (1968) "Kinetics ofRenaturation of DNA," J. Mol. Biol., 31:349-370; and Kanehisa, M. (1984)Nuc. Acids Res., 12:203-213.

E. Detection; VLSIPS™ Technology Scanning

Detection methods will be selected which are appropriate for theselected label. The scanning device need not necessarily be digitized orplaced into a specific digital database, though such would most likelybe done. For example, the analysis in fingerprinting could bephotographic. Where a standardized fingerprint substrate matrix is used,the pattern of hybridizations may be spatially unique and may becompared photographically. In this manner, each sample may have acharacteristic pattern of interactions and the likelihood of identicalpatterns will preferably be such low frequency that the fingerprintpattern indeed becomes a characteristic pattern virtually as unique asan individual's fingertip fingerprint. With a standardized substrate,every individual could be, in theory, uniquely identifiable on the basisof the pattern of hybridizing to the substrate.

Of course, the VLSIPS™ Technology scanning apparatus may also be usefulto generate a digitized version of the fingerprint pattern. In this way,the identification pattern can be provided in a linear string of digits.This sequence could also be used for a standardized identificationsystem providing significant useful medical transferability of specificdata. In one embodiment, the probes used are selected to be ofsufficiently high resolution to measure the antigens of the major histocompatibility complex. It might even be possible to providetransplantation matching data in a linear stream of data. Thefingerprinting data may provide a condensed version, or summary, of thelinear genetic data, or any other information data base.

F. Analysis

The analysis of the fingerprint will often be much simpler than a totalsequence determination. However, there may be particular types ofanalysis which will be substantially simplified by a selected group ofprobes. For example, probes which exhibit particular populationalheterogeneity may be selected. In this way, analysis may be simplifiedand practical utility enhanced merely by careful selection of thespecific probes and a careful matrix layout of those probes.

G. Substrate Reuse

As with the sequencing application, the fingerprinting usages may alsotake advantage of the reusability of the substrate. In this way, theinteractions can be disrupted, the substrate treated, and the renewedsubstrate is equivalent to an unused substrate.

H. Non-polynucleotide Aspects

Besides polynucleotide applications, the fingerprinting analysis may beapplied to other polymers, especially polypeptides, carbohydrates, andother polymers, both organic and inorganic. Besides using thefingerprinting method for analyzing a particular polymer, thefingerprinting method may be used to characterize various samples. Forexample, a cell or population of cells may be tested for theirexpression of specific antigens or their mRNA sequence intent. Forexample, a T-cell may be classified by virtue of its combination ofexpressed surface antigens. With specific reagents which interact withthese antigens, a cell or a population of cells or a lysed cell may beexposed to a VLSIPS substrate. The biological sample may be classifiedor characterized by analyzing the pattern of specific interaction. Thismay be applicable to a cell or tissue type, to the messenger RNApopulation expressed by a cell to the genetic content of a cell, or tovirtually any sample which can be classified and/or identified by itscombination of specific molecular properties.

The ability to generate a high density means for screening the presenceor absence of specific interactions allows for the possibility ofscreening for, if not saturating, all of a very large number of possibleinteractions. This is very powerful in providing the means for testingthe combinations of molecular properties which can define a class ofsamples. For example, a species of organism may be characterized by itsDNA sequences, e.g., a genetic fingerprint. By using a fingerprintingmethod, it may be determined that all members of that species aresufficiently similar in specific sequences that they can be easilyidentified as being within a particular group. Thus, newly definedclasses may be resolved by their similarity in fingerprint patterns.Alternatively, a non-member of that group will fail to share those manyidentifying characteristics. However, since the technology allowstesting of a very large number of specific interactions, it alsoprovides the ability to more finely distinguish between closely relateddifferent cells or samples. This will have important applications indiagnosing viral, bacterial, and other pathological on nonpathologicalinfections.

In particular, cell classification may be defined by any of a number ofdifferent properties. For example, a cell class may be defined by itsDNA sequences contained therein. This allows species identification forparasitic or other infections. For example, the human cell is presumablygenetically distinguishable from a monkey cell, but different humancells will share many genetic markers. At higher resolution, eachindividual human genome will exhibit unique sequences that can define itas a single individual.

Likewise, a developmental stage of a cell type may be definable by itspattern of expression of messenger RNA. For example, in particularstages of cells, high levels of ribosomal RNA are found whereasrelatively low levels of other types of messenger RNAs may be found. Thehigh resolution distinguishability provided by this fingerprintingmethod allows the distinction between cells which have relatively minordifferences in its expressed mRNA population. Where a pattern is shownto be characteristic of a stage, a stage may be defined by thatparticular pattern of messenger RNA expression.

In a similar manner, the antigenic determinants found on a protein mayvery well define the cell class. For example, immunological T-cells aredistinguishable from B-cells because, in part, the cell surface antigenson the cell types are distinguishable. Different T-cell subclasses canbe also distinguished from one another by whether they containparticular T-cell antigens. The present invention provides thepossibility for high resolution testing of many different interactionssimultaneously, and the definition of new cell types will be possible.

The high resolution VLSIPS™ substrate may also be used as a verypowerful diagnostic tool to test the combination of presence, of aplurality of different assays from a biological sample. For example, acancerous condition may be indicated by a combination of variousdifferent properties found in the blood. For example, a cancerouscondition may be indicated by a combination of expression of varioussoluble antigens found in the blood along with a high number of variouscellular antigens found on lymphocytes and/or particular celldegradation products. With a substrate as provided herein, a largenumber of different features can be simultaneously performed on abiological sample. In fact, the high resolution of the test will allowmore complete characterization of parameters which define particulardiseases. Thus, the power of diagnostic tests may be limited by theextent of statistical correlation with a particular condition ratherthan with the number of antigens or interactions which are tested. Thepresent invention provides the means to generate this large universe ofpossible reagents and the ability to actually accumulate thatcorrelative data.

In another embodiment, a substrate as provided herein may be used forgenetic screening. This would allow for simultaneous screening ofthousands of genetic markers. As the density of the matrix is increased,many more molecules can be simultaneously tested. Genetic screening thenbecomes a simpler method as the present invention provides the abilityto screen for thousands, tens of thousands, and hundreds of thousands,even millions of different possible genetic features. However, thenumber of high correlation genetic markers for conditions numbers onlyin the hundreds. Again, the possibility for screening a large number ofsequences provides the opportunity for generating the data which canprovide correlation between sequences and specific conditions orsusceptibility. The present invention provides the means to generateextremely valuable correlations useful for the genetic detection of thecausative mutation leading to medical conditions. In still anotherembodiment, the present invention would be applicable to distinguishingtwo individuals having identical genetic compositions. The antibodypopulation within an individual is dependent both on genetic andhistorical factors. Each individual experiences a unique exposure tovarious infectious agents, and the combined antibody expression ispartly determined thereby. Thus, individuals may also be fingerprintedby their immunological content, either of actively expressed antibodies,or their immunological memory. Similar sorts of immunological andenvironmental histories may be useful for fingerprinting, perhaps incombination with other screening properties. In particular, the presentinvention may be useful for screening allergic reactions orsusceptibilities, and a simple IgE specificity test may be useful indetermining a spectrum of allergies.

With the definition of new classes of cells, a cell sorter will be usedto purify them. Moreover, new markers for defining that class of cellswill be identified. For example, where the class is defined by its RNAcontent, cells may be screened by antisense probes which detect thepresence or absence of specific sequences therein. Alternatively, celllysates may provide information useful in correlating intracellularproperties with extracellular markers which indicate functionaldifferences. Using standard cell sorter technology with a fluorescenceor labeled antisense probe which recognizes the internal presence of thespecific sequences of interest, the cell sorter will be able to isolatea relatively homogeneous population of cells possessing the particularmarker. Using successive probes the sorting process should be able toselect for cells having a combination of a large number of differentmarkers.

In a non-polynucleotide embodiment, cells may be defined by the presenceof other markers. The markers may be carbohydrates, proteins, or othermolecules. Thus, a substrate having particular specific reagents, e.g.,antibodies, attached to it should be able to identify cells havingparticular patterns of marker expression. Of course, combinations ofthese made be utilized and a cell class may be defined by a combinationof its expressed mRNA, its carbohydrate expression, its antigens, andother properties. This fingerprinting should be useful in determiningthe physiological state of a cell or population of cells.

Having defined a cell type whose function or properties are defined bythe reagents attachable to a VLSIPS substrate, such as cellularantigens, these structural manifestations of function may be used tosort cells to generate a relatively homogeneous population of that classof cells. Standard cell sorter technology may be applied to purify sucha population, see, e.g., Dangl, J. and Herzenberg (1982) "Selection ofhybridomas and hybridoma variants using the fluorescence activated cellsorter," J. Immunological Methods 52:1-14; and Becton Dickinson,Fluorescence Activated Cell Sorter Division, San Jose, Calif., andCoulter Diagnostics, Hialeah, Fla.

With the fingerprinting method an identification means arises frommosaicism problems in an organism. A mosaic organism is one whosegenetic content in different cells is significantly different. Variousclonal populations should have similar genetic fingerprints, thoughdifferent clonal populations may have different genetic contents. See,for example, Suzuki et al. An Introduction to Genetic Analysis (4thEd.), Freeman and Co., New York, which is hereby incorporated herein byreference. However, this problem should be a relatively rare problem andcould be more carefully evaluated with greater experience using thefingerprinting methods.

The invention will also find use in detecting changes, both genetic andantigenic, e.g., in a rapidly "evolving" protozoa infection, orsimilarly changing organism.

V. MAPPING

A. General

The use of the present invention for mapping parallels its use forfingerprinting and sequencing. Where a polymer is a linear molecule, themapping provides the ability to locate particular segments along thelength of the polymer. Branched polymers can be treated as a series ofindividual linear polymers. The mapping provides the ability to locate,in a relative sense, the order of various subsequences. This may beachieved using at least two different approaches.

The first approach is to take the large sequence and fragment it atspecific points. The fragments are then ordered and attached to a solidsubstrate. For example, the clones resulting from a chromosome walkingprocess may be individually attached to the substrate by methods, e.g.,caged biotin techniques, indicated earlier. Segments of unknown mapposition will be exposed to the substrate and will hybridize to thesegment which contains that particular sequence. This procedure allowsthe rapid determination of a number of different labeled segments, eachmapping requiring only a single hybridization step once the substrate isgenerated. The substrate may be regenerated by removal of theinteraction, and the next mapping segment applied.

In an alternative method, a plurality of subsequences can be attached toa substrate. Various short probes may be applied to determine whichsegments may contain particular overlaps. The theoretical basis and adescription of this mapping procedure is contained in, e.g., Evans etal. 1989 "Physical Mapping of Complex Genomes by Cosmid MultiplexAnalysis," Proc. Natl. Acad. Sci. USA 86:5030-5034, and other referencescited above in the Section labeled "Overall Description." Using thisapproach, the details of the mapping embodiment are very similar tothose used in the fingerprinting embodiment.

B. Preparation of Substrate Matrix

The substrate may be generated in either of the methods generallyapplicable in the sequencing and fingerprinting embodiments. Thesubstrate may be made either synthetically, or by attaching otherwisepurified probes or sequences to the matrix. The probes or sequences maybe derived either from synthetic or biological means. As indicatedabove, the solid phase substrate synthetic methods may be utilized togenerate a matrix with positionally defined sequences. In the mappingembodiment, the importance of saturation of all possible subsequences ofa preselected length is far less important than in the sequencingembodiment, but the length of the probes used may be desired to be muchlonger. The processes for making a substrate which has longeroligonucleotide probes should not be significantly different from thosedescribed for the sequencing embodiments, but the optimizationparameters may be modified to comply with the mapping needs.

C. Labeling

The labeling methods will be similar to those applicable in sequencingand fingerprinting embodiments. Again, it may be desirable to fragmentthe target sequences.

D. Hybridization/Specific Interaction

The specificity of interaction between the targets and probe wouldtypically be closer to those used for fingerprinting embodiments, wherehomology is more important than absolute distinguishability of highfidelity complementary hybridization. Usually, the hybridizationconditions will be such that merely homologous segments will interactand provide a positive signal. Much like the fingerprinting embodiment,it may be useful to measure the extent of homology by successiveincubations at higher stringency conditions. Or, a plurality ofdifferent probes, each having various levels of homology may be used. Ineither way, the spectrum of homologies can be measured.

Where non-nucleic acid hybridization is involved, the specificinteractions may also be compared in a fingerprint-like manner. Thespecific reagents may have less specificity, e.g., monoclonal antibodieswhich recognize a broader spectrum of sequences may be utilized relativeto a sequencing embodiment. Again, the specificity of interaction may bemeasured under various conditions of increasing stringency to determinethe spectrum of matching across the specific probes selected, or anumber of different stringency reagents may be included to indicate thebinding affinity.

E. Detection

The detection methods used in the mapping procedure will be virtuallyidentical to those used in the fingerprinting embodiment. The detectionmethods will be selected in combination with the labeling methods.

F. Analysis

The analysis of the data in a mapping embodiment will typically besomewhat different from that in fingerprinting. The fingerprintingembodiment will test for the presence or absence of specific orhomologous segments. However, in the mapping embodiment, the existenceof an interaction is coupled with some indication of the location of theinteraction. The interaction is mapped in some manner to the physicalpolymer sequence. Some means for determining the relative positions ofdifferent probes is performed. This may be achieved by synthesis of thesubstrate in pattern, or may result from analysis of sequences afterthey have been attached to the substrate.

For example, the probes may be randomly positioned at various locationson the substrate. However, the relative positions of the variousreagents in the original polymer may be determined by using shortfragments, e.g., individually, as target molecules which determine theproximity of different probes. By an automated system of testing eachdifferent short fragment of the original polymer, coupled with properanalysis, it will be possible to determine which probes are adjacent oneanother on the original target sequence and correlate that withpositions on the matrix. In this way, the matrix is useful fordetermining the relative locations of various new segments in theoriginal target molecule. This sort of analysis is described in Evans,and the related references described above.

G. Substrate Reuse

The substrate should be reusable in the manner described in thefingerprinting section. The substrate is renewed by removal of thespecific interactions and is washed and prepared for successive cyclesof exposure to new target sequences.

H. Non-polynucleotide Aspects

The mapping procedure may be used on other molecules thanpolynucleotides. Although hybridization is one type of specificinteraction which is clearly useful for use in this mapping embodiment,antibody reagents may also be very useful. In the same way thatpolypeptide sequencing or other polymers may be sequenced by thereagents and techniques described in the sequencing section andfingerprinting section, the mapping embodiment may also be usedsimilarly.

In another form of mapping, as described above in the fingerprintingsection, the developmental map of a cell or biological system may bemeasured using fingerprinting type technology. Thus, the mapping may bealong a temporal dimension rather than along a polymer dimension Themapping or fingerprinting embodiments may also be used in determiningthe genetic rearrangements which may be genetically important, as inlymphocyte and B-cell development. In another example, variousrearrangements or chromosomal dislocations may be tested by either thefingerprinting or mapping methods. These techniques are similar in manyrespects and the fingerprinting and mapping embodiments may overlap inmany respects.

VI. ADDITIONAL SCREENING AND APPLICATIONS

A. Specific Interactions

As originally indicated in the parent filing of VLSIPS™ Technology, theproduction of a high density plurality of spatially segregated polymersprovides the ability to generate a very large universe or repertoire ofindividually and distinct sequence possibilities. As indicated above,particular oligonucleotides may be synthesized in automated fashion atspecific locations on a matrix. In fact, these oligonucleotides may beused to direct other molecules to specific locations by linking specificoligonucleotides to other reagents which are in batch exposed to thematrix and hybridized in a complementary fashion to only those locationswhere the complementary oligonucleotide has been synthesized on thematrix. This allows for spatially attaching a plurality of differentreagents onto the matrix instead of individually attaching each separatereagent at each specific location. Although the caged biotin methodallows automated attachment, the speed of the caged biotin attachmentprocess is relatively slow and requires a separate reaction for eachreagent being attached. By use of the oligonucleotide method, thespecificity of position can be done in an automated and parallelfashion. As each reagent is produced, instead of directly attaching eachreagent at each desired position, the reagent may be attached to aspecific desired complementary oligonucleotide which will ultimately bespecifically directed toward locations on the matrix having acomplementary oligonucleotide attached thereat.

In addition, the technology allows screening for specificity ofinteraction with particular reagents. For example, the oligonucleotidesequence specificity of binding of a potential reagent may be tested bypresenting to the reagent all of the possible subsequences available forbinding. Although secondary or higher order sequence specific featuresmight not be easily screenable using this technology, it does provide aconvenient, simple, quick, and thorough screen of interactions between areagent and its target recognition sequences. See, e.g., Pfeifer et al.(1989) Science 246:810-812.

For example, the interaction of a promoter protein with its targetbinding sequence may be tested for many different, or all, possiblebinding sequences. By testing the strength of interactions under variousdifferent conditions, the interaction of the promoter protein with eachof the different potential binding sites may be analyzed. The spectrumof strength of interactions with each different potential binding sitemay provide significant insight into the types of features which areimportant in determining specificity.

An additional example of a sequence specific interaction betweenreagents is the testing of binding of a double stranded nucleic acidstructure with a single stranded oligonucleotide. Often, a triplestranded structure is produced which has significant aspects of sequencespecificity. Testing of such interactions with either sequencescomprising only natural nucleotides, or perhaps the testing ofnucleotide analogs may be very important in screening for particularlyuseful diagnostic or therapeutic reagents. See, e.g., Haner and Dervan(1990) Biochemistry 29:9761-6765, and references therein.

B. Sequence Comparisons

Once a gene is sequenced, the present invention provides a means tocompare alleles or related sequences to locate and identify differencesfrom the control sequence. This would be extremely useful in furtheranalysis of genetic variability at a specific gene locus.

C. Categorizations

As indicated above in the fingerprinting and mapping embodiments, thepresent invention is also useful in defining specific stages in thetemporal sequence of cells, e.g., development, and the resulting tissueswithin an organism. For example, the developmental stage of a cell, orpopulation of cells, can be dependent upon the expression of particularmessenger RNAs or cellular antigens. The screening procedures providedallow for high resolution definition of new classes of cells. Inaddition, the temporal development of particular cells will becharacterized by the presence or expression of various mRNAs. Means tosimultaneously screen a plurality or very large number of differentsequences are provided. The combination of different markers madeavailable dramatically increases the ability to distinguish fairlyclosely related cell types. Other markers may be combined with markersand methods made available herein to define new classifications ofbiological samples, e.g., based upon new combinations of markers.

The presence or absence of particular marker sequences will be used todefine temporal developmental stages. Once the stages are defined,fairly simple methods can be applied to actually purify those particularcells. For example, antisense probes or recognition reagents may be usedwith a cell sorter to select those cells containing or expressing thecritical markers. Alternatively, the expression of those sequences mayresult in specific antigens which may also be used in defining cellclasses and sorting those cells away from others. In this way, forexample, it should be possible to select a class of omnipotent immunesystem cells which are able to completely regenerate a human immunesystem. Based upon the cellular classes defined by the parameters madeavailable by this technology, purified classes of cells havingidentifiable differences, structural or functional, are made available.

In an alternative embodiment, a plurality of antigens or specificbinding proteins attached to the substrate may be used to defineparticular cell types. For example, subclasses of T-cells are defined,in part, by the combination of expressed cell surface antigens. Thepresent invention allows for the simultaneous screening of a largeplurality of different antigens together. Thus, higher resolutionclassification of different T-cell subclasses becomes possible and, withthe definitions and functional differences which correlate with thoseantigenic or other parameters, the ability to purify those cell typesbecomes available. This is applicable not only to T-cells, but also tolymphocyte cells, or even to freely circulating cells. Many of the cellsfor which this would be most useful will be immobile cells found inparticular tissues or organs. Tumor cells will be diagnosed or detectedusing these fingerprinting techniques. Coupled with a temporal change instructure, developmental classes may also be selected and defined usingthese technologies. The present invention also provides the ability notonly to define new classes of cells based upon functional or structuraldifferences, but it also provides the ability to select or purifypopulations of cells which share these particular properties. Standardcell sorting procedures using antibody markers may be used to detectextracellular features. Intracellular features would also be detectableby introducing the label reagents into the cell. In particular,antisense DNA or RNA molecules may be introduced into a cell to detectRNA sequences therein. See, e.g., Weintraub (1990) Scientific American262:40-46.

D. Statistical Correlations

In an additional embodiment, the present invention also allows for thehigh resolution correlation of medical conditions with various differentmarkers. For example, the presently available technology, when appliedto amniocentesis or other genetic screening methods, typically screensfor tens of different markers at most. The present invention allowssimultaneous screening for tens, hundreds, thousands, tens of thousands,hundreds of thousands, and even millions of different genetic sequences.Thus, applying the fingerprinting methods of the present invention to asufficiently large population allows detailed statistical analysis to bemade, thereby correlating particular medical conditions with particularmarkers, typically antigenic or genetic. Tumor specific antigens will beidentified using the present invention.

Various medical conditions may be correlated against an enormous database of the sequences within an individual. Genetic propensities andcorrelations then become available and high resolution geneticpredictability and correlation become much more easily performed. Withthe enormous data base, the reliability of the predictions is alsobetter tested. Particular markers which are partially diagnostic ofparticular medical conditions or medical susceptibilities will beidentified and provide direction in further studies and more carefulanalysis of the markers involved. Of course, as indicated above in thesequencing embodiment, the present invention will find much use inintense sequencing projects. For example, sequencing of the entire humangenome in the human genome project will be greatly simplified andenabled by the present invention.

VI. FORMATION OF SUBSTRATE

The substrate is provided with a pattern of specific reagents which arepositionally localized on the surface of the substrate. This matrix ofpositions is defined by the automated system which produces thesubstrate. The instrument will typically be one similar to thatdescribed in Pirrung et al. (1992) U.S. Pat. No. 5,143,854, and Ser. No.07/624,120, now abandoned. The instrumentation described therein isdirectly applicable to the applications used here. In particular, theapparatus comprises a substrate, typically a silicon containingsubstrate, on which positions on the surface may be defined by acoordinate system of positions. These positions can be individuallyaddressed or detected by the VLSIPS™ Technology apparatus.

Typically, the VLSIPS™ Technology apparatus uses optical methods used insemiconductor fabrication applications. In this way, masks may be usedto photo-activate positions for attachment or synthesis of specificsequences on the substrate. These manipulations may be automated by thetypes of apparatus described in Pirrung et al. (1992) U.S. Pat. No.5,143,854 and Ser. No. 07/624,120, now abandoned.

Selectively removable protecting groups allow creation of well definedareas of substrate surface having differing reactivities. Preferably,the protecting groups are selectively removed from the surface byapplying a specific activator, such as electromagnetic radiation of aspecific wavelength and intensity. More preferably, the specificactivator exposes selected areas of surface to remove the protectinggroups in the exposed areas.

Protecting groups of the present invention are used in conjunction withsolid phase oligomer syntheses, such as peptide syntheses using naturalor unnatural amino acids, nucleotide syntheses using deoxyribonucleicand ribonucleic acids, oligosaccharide syntheses, and the like. Inaddition to protecting the substrate surface from unwanted reaction, theprotecting groups block a reactive end of the monomer to preventself-polymerization. For instance, attachment of a protecting group tothe amino terminus of an activated amino acid, such as theN-hydroxysuccinimide-activated ester of the amino acid prevents theamino terminus of one monomer from reacting with the activated esterportion of another during peptide synthesis.

Alternatively, the protecting group may be attached to the carboxylgroup of an amino acid to prevent reaction at this site. Most protectinggroups can be attached to either the amino or the carboxyl group of anamino acid, and the nature of the chemical synthesis will dictate whichreactive group will require a protecting group. Analogously, attachmentof a protecting group to the 5'-hydroxyl group of a nucleoside duringsynthesis using for example, phosphate-triester coupling chemistry,prevents the 5'-hydroxyl of one nucleoside from reacting with the3'-activated phosphate-triester of another.

Regardless of the specific use, protecting groups are employed toprotect a moiety on a molecule from reacting with another reagent.Protecting groups of the present invention have the followingcharacteristics: they prevent selected reagents from modifying the groupto which they are attached; they are stable (that is, they remainattached) to the synthesis reaction conditions; they are removable underconditions that do not adversely affect the remaining structure; andonce removed, do not react appreciably with the surface or surface-boundoligomer. The selection of a suitable protecting group will depend, ofcourse, on the chemical nature of the monomer unit and oligomer, as wellas the specific reagents they are to protect against.

In a preferred embodiment, the protecting groups will bephotoactivatable. The properties and uses of photoreactive protectingcompounds have been reviewed. See, McCray et al., Ann. Rev. of Biophys.and Biophys. Chem. (1989) 18:239-270, which is incorporated herein byreference. Preferably, the photosensitive protecting groups will beremovable by radiation in the ultraviolet (UV) or visible portion of theelectromagnetic spectrum. More preferably, the protecting groups will beremovable by radiation in the near UV or visible portion of thespectrum. In some embodiments, however, activation may be performed byother methods such as localized heating, electron beam lithography,laser pumping, oxidation or reduction with microelectrodes, and thelike. Sulfonyl compounds are suitable reactive groups for electron beamlithography. Oxidative or reductive removal is accomplished by exposureof the protecting group to an electric current source, preferably usingmicroelectrodes directed to the predefined regions of the surface whichare desired for activation. A more detailed description of theseprotective groups is provided in Ser. No. 07/624,120, now abandoned,which is hereby incorporated herein by reference.

The density of reagents attached to a silicon substrate may be varied bystandard procedures. The surface area for attachment of reagents may beincreased by modifying the silicon surface. For example, a matte surfacemay be machined or etched on the substrate to provide more sites forattachment of the particular reagents. Another way to increase thedensity of reagent binding sites is to increase the derivitizationdensity of the silicon. Standard procedures for achieving this aredescribed, below.

One method to control the derivatization density is to highly derivatizethe substrate with photochemical groups at high density. The substrateis then photolyzed for various predetermined times, which photoactivatethe groups at a measurable rate, and react them with a capping reagent.By this method, the density of linker groups may be modulated by using adesired time and intensity of photoactivation.

In many applications, the number of different sequences which may beprovided may be limited by the density and the size of the substrate onwhich the matrix pattern is generated. In situations where the densityis insufficiently high to allow the screening of the desired number ofsequences, multiple substrates may be used to increase the number ofsequences tested. Thus, the number of sequences tested may be increasedby using a plurality of different substrates. Because the VLSIPSapparatus is almost fully automated, increasing the number of substratesdoes not lead to a significant increase in the number of manipulationswhich must be performed by humans. This again leads to greaterreproducibility and speed in the handling of these multiple substrates.

A. Instrumentation

The concept of using VLSIPS™ Technology generally allows a pattern or amatrix of reagents to be generated. The procedure for making the patternis performed by any of a number of different methods. An apparatus andinstrumentation useful for generating a high density VLSIPS substrate isdescribed in detail in Pirrung et al. (1992) U.S. Pat. No. 5,143,854 andSer. No. 07/624,120, now abandoned.

B. Binary Masking

The details of the binary masking are described in an accompanyingapplication filed simultaneously with this, Ser. No. 07/624,120, nowabandoned, whose specification is incorporated herein by reference.

For example, the binary masking technique allows for producing aplurality of sequences based on the selection of either of twopossibilities at any particular location. By a series of binary maskingsteps, the binary decision may be the determination, on a particularsynthetic cycle, whether or not to add any particular one of thepossible subunits. By treating various regions of the matrix pattern inparallel, the binary masking strategy provides the ability to carry outspatially addressable parallel synthesis.

C. Synthetic Methods

The synthetic methods in making a substrate are described in the parentapplication, Pirrung et al. (1992) U.S. Pat. No. 5,143,854. Theconstruction of the matrix pattern on the substrate. will typically begenerated by the use of photosensitive reagents. By use ofphoto-lithographic optical methods, particular segments of the substratecan be irradiated with light to activate or deactivate blocking agents,e.g., to protect or deprotect particular chemical groups. By anappropriate sequence of photo-exposure steps at appropriate times withappropriate masks and with appropriate reagents, the substrates can haveknown polymers synthesized at positionally defined regions on thesubstrate. Methods for synthesizing various substrates are described inPirrung et al. (1992) U.S. Pat. No. 5,143,854 and Ser. No. 07/624,120,now abandoned. By a sequential series of these photo-exposure andreaction manipulations, a defined matrix pattern of known sequences maybe generated, and is typically referred to as a VLSIPSTM Technologysubstrate. In the nucleic acid synthesis embodiment, nucleosides used inthe synthesis of DNA by photolytic methods will typically be one of thetwo forms shown below: ##STR1##

B=Adenine, Cytosine, Guanine, or Thymine

In I, the photolabile group at the 5' position is abbreviated NV(nitroveratryl) and in II, the group is abbreviated NVOC (nitroveratryloxycarbonyl). Although not shown in FIG. C, the bases (adenine,cytosine, and guanine) contain exocyclic NH₂ groups which must beprotected during DNA synthesis. Thymine contains no exocyclic NH₂ andtherefore requires no protection. The standard protecting groups forthese amines are shown below: ##STR2##

Other amides of the general formula ##STR3## where R may be alkyl oraryl have been used.

Another type of protecting group FMOC (9-fluorenyl methoxycarbonyl) iscurrently being used to protect the exocyclic amines of the three bases:##STR4##

The advantage of the FMOC group is that it is removed under mildconditions (dilute organic bases) and can be used for all three bases.The amide protecting groups require more Nucleosides used as 5'--OHprobes, useful in verifying correct VLSIPS synthetic function, include,for example, the following: ##STR5##

These compounds are used to detect where on a substrate photolysis hasoccurred by the attachment of either III or V to the newly generated5'--OH. In the case of III, after the phosphate attachment is made, thesubstrate is treated with a dilute base to remove the FMOC group. Theresulting amine can be reacted with FITC and the substrate examined byfluorescence microscopy. This indicates the proper generation of a5'--OH. In the case of compound IV, after the phosphate attachment ismade, the substrate is treated with FITC labeled streptavidin and thesubstrate again may be examined by fluorescence microscopy. Otherprobes, although not nucleoside based, have included the following:##STR6##

The method of attachment of the first nucleoside to he surface of thesubstrate depends on the functionality of he groups at the substratesurface. If the surface is amine unctionalized, an amide bond is made(see example below) ##STR7##

If the surface is hydroxy functionalized, a phosphate nd is made (seeexample below): ##STR8##

In both cases, the thymidine example is illustrated, any one of the fourphosphoramidite activated nucleosides be used in the first step.

Photolysis of the photolabile group NV or NVOC on the 5' positions ofthe nucleosides is carried out at .sup.˜ 362 nm with an intensity of 14mW/cm² for 10 minutes with the substrate side (side containing thephotolabile group) immersed in dioxane. After the coupling of the nextnucleoside is complete, the photolysis is repeated followed by anothercoupling until the desired oligomer is obtained.

One of the most common 3'-O-protecting groups is the ester, inparticular the acetate: ##STR9##

The groups can be removed by mild base treatment 0.1N NaOH/MeOH or K₂CO₃ /H₂ O/MeOH.

Another group used most often is the silyl ether: ##STR10##

These croups can be removed by neutral conditions using 1 Mtetra-n-butylammonium fluoride in THF or under acid conditions.

With respect to photodeprotection, the nitroveratryl group could also beused to protect the 3'-position. ##STR11##

Here, light (photolysis) would be used to remove these protectinggroups.

A variety of ethers can also be used in the protection of the3'-O-position: ##STR12##

Removal of these groups usually involves acid or catalytic methods.

Note that corresponding linkages and photoblocked amino acids aredescribed in detail in Ser. No. 07/624,120, now abandoned, which ishereby incorporated herein by reference.

Although the specificity of interactions at particular locations willusually be homogeneous due to a homogeneous polymer being synthesized ateach defined location, for certain purposes, it may be useful to havemixed polymers with a commensurate mixed collection of interactionsoccurring at specific defined locations, or degeneracy reducinganalogues, which have been discussed above and show broad specificity inbinding. Then, a positive interaction signal may result from any of anumber of sequences contained therein.

As an alternative method of generating a matrix pattern on a substrate,preformed polymers may be individually attached at particular sites onthe substrate. This may be performed by individually attaching reagentsone at a time to specific positions on the matrix, a process which maybe automated. See, e.g., Ser. No. 07/435,316, now abandoned, and Barrettet al. (1993) U.S. Pat. No. 5,252,743. Another way of generating apositionally defined matrix pattern on a substrate is to haveindividually specific reagents which interact with each specificposition on the substrate. For example, oligonucleotides may besynthesized at defined locations on the substrate. Then the substratewould have on its surface a plurality of regions having homogeneousoligonucleotides attached at each position.

In particular, at least four different substrate preparation proceduresare available for treating a substrate surface. They are the standardVLSIPS™ Technology method, polymeric substrates, Durapore , andsynthetic beads or fibers. The treatment labeled "standard VLSIPS™Technology" method is described in Ser. No. 07/624,120, now abandoned,and involves applying amino-propyltriethoxysilane to a glass surface.

The polymeric substrate approach involves either of two ways ofgenerating a polymeric substrate. The first uses a high concentration ofaminopropyltriethoxysilane (2-20%) in an aqueous ethanol solution (95%).This allows the silane compound to polymerize both in solution and onthe substrate surface, which provides a high density of amines on thesurface of the glass. This density is contrasted with the standardVLSIPS method. This polymeric method allows for the deposition on thesubstrate surface of a monolayer due to the anhydrous method used withthe aforementioned silane.

The second polymeric method involves either the coating or covalentbinding of an appropriate acrylic acid polymer onto the substratesurface. In particular, e.g., in DNA synthesis, a monomer such as ahydroxypropylacrylate is used to generate a high density of hydroxylgroups on the substrate surface, allowing for the formation of phosphatebonds. An example of such a compound is shown: ##STR13##

The method using a Durapore™ membrane (Millipore) consists of apolyvinylidine difluoride coating with crosslinked polyhydroxylpropylacrylate PVDF-HPA!: ##STR14## Here the building up of, e.g., a DNAoligomer, can be started immediately since phosphate bonds to thesurface can be accomplished in the first step with no need formodification. A nucleotide dimer (5'-C-T-3') has been successfully madeon this substrate.

The fourth method utilizes synthetic beads or fibers. This would useanother substrate, such as a teflon copolymer graft bead or fiber, whichis covalently coated with an organic layer (hydrophilic) terminating inhydroxyl sites (commercially available from Molecular Biosystems, Inc.)This would offer the same advantage as the Durapore™ membrane, allowingfor immediate phosphate linkages, but would give additional contour bythe 3-dimensional growth of oligomers.

A matrix pattern of new reagents may be targeted to each specificoligonucleotide position by attaching a complementary oligonucleotide towhich the substrate bound form is complementary. For instance, a numberof regions may have homogeneous oligonucleotides synthesized at variouslocations. Oligonucleotide sequences complementary to each of these canbe individually generated and linked to a particular specific reagents.Often these specific reagents will be antibodies. As each of these isspecific for finding its complementary oligonucleotide, each of thespecific reagents will bind through the oligonucleotide to theappropriate matrix position. A single step having a combination ofdifferent specific reagents being attached specifically to a particularoligonucleotide will thereby bind to its complement at the definedmatrix position. The oligonucleotides will typically then be covalentlyattached, using, e.g., an acridine dye, for photocrosslinking. Psoralenis a commonly used acridine dye for photocrosslinking purposes, see,e.g., Song et al. (1979) Photochem. Photobiol. 29:1177-1197; Cimino etal. (1985) Ann. Rev. Biochem. 54:1151-1193; Parsons (1980) Photochem.Photobiol. 32:813-821; and Dattagupta et al. (1985) U.S. Pat. No.4,542,102, and (1987) U.S. Pat. No. 4,713,326; each of which is herebyincorporated herein by reference. This method allows a single attachmentmanipulation to attach all of the specific reagents to the matrix atdefined positions and results in the specific reagents beinghomogeneously located at defined positions. In many embodiments, thespecific reagents will be antibodies.

In an alternative embodiment, antibody molecules may be used tospecifically direct binding to defined positions on a substrate. TheVLSIPS technology may be used to generate specific epitopes at eachposition on the substrate. Antibody molecules having specificity ofinteraction may be used to attach oligonucleotides, thereby avoiding theinterference of internal polynucleotide sequences from binding to thesubstrate complementary oligonucleotides. In fact, the specificity ofinteraction for positional targeting may be achieved by use ofnucleotide analogues which do not interact with the natural nucleotides.For example, other synthetic nucleotides have been made which undergobase pairing, thereby providing the specificity of targeting, but thesynthetic nucleotides also do not interact with the natural biologicalnucleotides. Thus, synthetic oligonucleotides would be useful forattachment to biological nucleotides and specific targeting. Moreover,the VLSIPS synthetic processes would be useful in generating the VLSIPSsubstrate, and standard oligonucleotide synthesis could be applied, withminor modifications, to produce the complementary sequences which wouldbe attached to other specific reagents.

D. Surface Immobilization

1. caged biotin

An alternative method of attaching reagents in a positionally definedmatrix pattern is to use a caged biotin system. See Barrett et al.(1993) U.S. Pat. No. 5,252,743, which is hereby incorporated herein byreference, for additional details on the chemistry and application ofcaged biotin embodiments. In short, the caged biotin has aphotosensitive blocking moiety which prevents the combination of avidinto biotin. At positions where the photo-lithographic process has removedthe blocking group, high affinity biotin sites are generated. Thus, by asequential series of photolithographic deblocking steps interspersedwith exposure of those regions to appropriate biotin containingreagents, only those locations where the deblocking takes place willform an avidin-biotin interaction. Because the avidin-biotin binding isvery tight, this will usually be virtually irreversible binding.

2. crosslinked interactions

The surface immobilization may also take place by photo crosslinking ofdefined oligonucleotides linked to specific reagents. Afterhybridization of the complementary oligonucleotides, theoligonucleotides may be crosslinked by a reagent by psoralen or anothersimilar type of acridine dye. Other useful cross linking reagents aredescribed in Dattagupta et al. (1985) U.S. Pat. No. 4,542,102, and(1987) U.S. Pat. No. 4,713,326.

In another embodiment, colony or phage plaque transfer of biologicalpolymers may be transferred directly onto a silicon substrate. Forexample, a colony plate may be transferred onto a substrate having ageneric oligonucleotide sequence which hybridizes to another genericcomplementary sequence contained on all of the vectors into whichinserts are cloned. This will specifically only bind those moleculeswhich are actually contained in the vectors containing the desiredcomplementary sequence. This immobilization allows for producing amatrix onto which a sequence specific reagent can bind, or for otherpurposes. In a further embodiment, a plurality of different vectors eachhaving a specific oligonucleotide attached to the vector may bespecifically attached to particular regions on a matrix having acomplementary oligonucleotide attached thereto.

VIII. HYBRIDIZATION/SPECIFIC INTERACTION

A. General

As discussed previously in the VLSIPS™ Technology parent applications,the VLSIPS™ technology substrates may be used for screening for specificinteractions with sequence specific targets or probes.

In addition, the availability of substrates having the entire repertoireof possible sequences of a defined length opens up the possibility ofsequencing by hybridization. This sequence may be de novo determinationof an unknown sequence, particularly of nucleic acid, verification of asequence determined by another method, or an investigation of changes ina previously sequenced gene, locating and identifying specific changes.For example, often Maxam and Gilbert sequencing techniques are appliedto sequences which have been determined by Sanger and Coulson. Each ofthose sequencing technologies have problems with resolving particulartypes of sequences. Sequencing by hybridization may serve as a third andindependent method for verifying other sequencing techniques. See, e.g.,(1988) Science 242:1245.

In addition, the ability to provide a large repertoire of particularsequences allows use of short subsequences and hybridization as a meansto fingerprint a sample. This may be used in a nucleic acid, as well asother polymer embodiments. For example, fingerprinting to a high degreeof specificity of sequence matching may be used for identifying highlysimilar samples, e.g., those exhibiting high homology to the selectedprobes. This may provide a means for determining classifications ofparticular sequences. This should allow determination of whetherparticular genomes of bacteria, phage, or even higher cells might berelated to one another.

In addition, fingerprinting may be used to identify an individual sourceof biological sample. See, e.g., Lander, E. (1989) Nature, 339:501-505,and references therein. For example, a DNA fingerprint may be used todetermine whether a genetic sample arose from another individual. Thiswould be particularly useful in various sorts of forensic tests todetermine, e.g., paternity or sources of blood samples. Significantdetail on the particulars of genetic fingerprinting for identificationpurposes are described in, e.g., Morris et al. (1989) "Biostatisticalevolution of evidence from continuous allele frequency distribution DNAprobes in reference to disputed paternity of identity," J. ForensicScience 34:1311-1317; and Neufeld et al. (1990) Scientific American262:46-53; each of which is hereby incorporated herein by reference.

In another embodiment, a fingerprinting-like procedure may be used forclassifying cell types by analyzing a pattern of specific nucleic acidspresent in the cell. A series of antibodies may be used to identify cellmarkers, e.g., proteins, usually on the cell surface, but intracellularmarkers may also be used. Antigens which are extracellularly expressedare preferred so cell lysis is unnecessary in the screening, butintracellular markers may also be useful. The markers will usually beproteins, but may be nucleic acids, lipids, metabolites, carbohydrates,or other cellular components. See, e.g., Winkelgren, I. (1990) ScienceNews 136:234-237, which indicates extracellular DNA may be common, andsuggesting that such might be characteristic of cell types, stage, orphysiology. This may also be useful in defining the temporal stage ofdevelopment of cells, e.g., stem cells or other cells which undergotemporal changes in development. For example, the stage of a cell, orgroup of cells, may be tested or defined by isolating a sample of mRNAfrom the population and testing to see what sequences are present inmessenger populations. Direct samples, or amplified samples, may beused. Where particular mRNA or other nucleic acid sequences may becharacteristic of or shown to be characteristic of particulardevelopmental stages, physiological states, or other conditions, thisfingerprinting method may define them. Similar sorts of fingerprintingmay be used for determining T-cell classes or perhaps even to generateclassification schemes for such proteins as major histocompatibilitycomplex antigens. Thus, the ability to make these substrates allows boththe generation of reagents which will be used for defining subclasses orclasses of cells or other biological materials, but also provides themechanisms for selecting those cells which may be found in definedpopulation groups.

In addition to cell classification defined by such a combination ofproperties, typically expression of extracellular antigens, the presentinvention also provides the means for isolating homogeneous populationof cells. Once the antigenic determinants which define a cell class havebeen identified, these antigens may be used in a sequential selectionprocess to isolate only those cells which exhibit the combination ofdefining structural properties.

The present invention may also be used for mapping sequences within alarger segment. This may be performed by at least two methods,particularly in reference to nucleic acids. Often, enormous segments ofDNA are subcloned into a large plurality of subsequences. Ordering thesesubsequences may be important in determining the overlaps of sequencesupon nucleotide determinations. Mapping may be performed by immobilizingparticularly large segments onto a matrix using the VLSIPS™ Technology.Alternatively, sequences may be ordered by virtue of subsequences sharedby overlapping segments. See, e.g., Craig et al. (1990) Nuc. Acids Res.18:2653-2660; Michiels et al. (1987) CABIOS 3:203-210; and Olson et al.(1986) Proc. Natl. Acad. Sci. USA 83:7826-7830.

B. Important Parameters

The extent of specific interaction between reagents immobilized to theVLSIPS™ Technology substrate and another sequence specific reagent maybe modified by the conditions of the interaction. Sequencing embodimentstypically require high fidelity hybridization and the ability todiscriminate perfect matching from imperfect matching. Fingerprintingand mapping embodiments may be performed using less stringentconditions, depending upon the circumstances.

For example, the specificity of antibody/antigen interaction may dependupon such parameters as pH, salt concentration, ionic composition,solvent composition, detergent composition and concentration, andchaotropic agent concentration. See, e.g., Harlow and Lane (1988)Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York. Bycareful control of these parameters, the affinity of binding may bemapped across different sequences.

In a nucleic acid hybridization embodiment, the specificity and kineticsof hybridization have been described in detail by, e.g., Wetmur andDavidson (1968) J. Mol. Biol., 31:349-370, Britten and Kohne (1968)Science 161:529-530, and Kanehisa, (1984) Nuc. Acids Res. 12:203-213,each of which is hereby incorporated herein by reference. Parameterswhich are well known to affect specificity and kinetics of reactioninclude salt conditions, ionic composition of the solvent, hybridizationtemperature, length of oligonucleotide-matching sequences, guanine andcytosine (GC) content, presence of hybridization accelerators, pH,specific bases found in the matching sequences, solvent conditions, andaddition of organic solvents.

In particular, the salt conditions required for driving highlymismatched sequences to completion typically include a high saltconcentration. The typical salt used is sodium chloride (NaCl), however,other ionic salts may be utilized, e.g., KCl. Depending on the desiredstringency hybridization, the salt concentration will often be less thanabout 3 molar, more often less than 2.5 molar, usually less than about 2molar, and more usually less than about 1.5 molar. For applicationsdirected towards higher stringency matching, the salt concentrationswould typically be lower. ordinary high stringency conditions willutilize salt concentration of less than about 1 molar, more often lessthen about 750 millimolar, usually less than about 500 millimolar, andmay be as low as about 250 or 150 millimolar.

The kinetics of hybridization and the stringency of hybridization bothdepend upon the temperature at which the hybridization is performed andthe temperature at which the washing steps are performed. Temperaturesat which steps for low stringency hybridization are desired wouldtypically be lower temperatures, e.g., ordinarily at least about 15° C.,more ordinarily at least about 20° C, usually at least about 25° C., andmore usually at least about 30° C. For those applications requiring highstringency hybridization, or fidelity of hybridization and sequencematching, temperatures at which hybridization and washing steps areperformed would typically be high. For example, temperatures in excessof about 35° C. would often be used, more often in excess of about 40°C., usually at least about 45° C., and occasionally even temperatures ashigh as about 50° C. or 60° C. or more. Of course, the hybridization ofoligonucleotides may be disrupted by even higher temperatures. Thus, forstripping of targets from substrates, as discussed below, temperaturesas high as 80° C., or even higher may be used.

The base composition of the specific oligonucleotides involved inhybridization affects the temperature of melting, and the stability ofhybridization as discussed in the above references. However, the bias ofGC rich sequences to hybridize faster and retain stability at highertemperatures can be compensated for by the inclusion in thehybridization incubation or wash steps of various buffers. Samplebuffers which accomplish this result include the triethly-and trimethylammonium buffers. See, e.g., Wood et al. (1987) Proc. Natl. Acad. Sci.USA, 82:1585-1588, and Khrapko, K. et al. (1989) FEBS Letters256:118-122.

The rate of hybridization can also be affected by the inclusion ofparticular hybridization accelerators. These hybridization acceleratorsinclude the volume exclusion agents characterized by dextran sulfate, orpolyethylene glycol (PEG). Dextran sulfate is typically included at aconcentration of between 1% and 40% by weight. The actual concentrationselected depends upon the application, but typically a fasterhybridization is desired in which the concentration is optimized for thesystem in question. Dextran sulfate is often included at a concentrationof between 0.5% and 2% by weight or dextran sulfate at a concentrationbetween about 0.5% and 5%. Alternatively, proteins which acceleratehybridization may be added, e.g., the recA protein found in E. coli orother homologous proteins.

With respect to those embodiments where specific reagents are notoligonucleotides, the conditions of specific interaction would depend onthe affinity of binding between the specific reagent and its target.Typically parameters which would be of particular importance would bepH, salt concentration anion and cation compositions, bufferconcentration, organic solvent inclusion, detergent concentration, andinclusion of such reagents such as chaotropic agents. In particular, theaffinity of binding may be tested over a variety of conditions bymultiple washes and repeat scans or by using reagents with differencesin binding affinity to determine which reagents bind or do not bindunder the selected binding and washing conditions. The spectrum ofbinding affinities may provide an additional dimension of informationwhich may be very useful in identification purposes and mapping.

Of course, the specific hybridization conditions will be selected tocorrespond to a discriminatory condition which provides a positivesignal where desired but fails to show a positive signal at affinitieswhere interaction is not desired. This may be determined by a number oftitration steps or with a number of controls which will be run duringthe hybridization and/or washing steps to determine at what point thehybridization conditions have reached the stage of desired specificity.

IX. DETECTION METHODS

Methods for detection depend upon the label selected. The criteria forselecting an appropriate label are discussed below, however, afluorescent label is preferred because of its extreme sensitivity andsimplicity. Standard labeling procedures are used to determine thepositions where interactions between a sequence and a reagent takeplace. For example, if a target sequence is labeled and exposed to amatrix of different probes, only those locations where probes dointeract with the target will exhibit any signal. Alternatively, othermethods may be used to scan the matrix to determine where interactiontakes place. Of course, the spectrum of interactions may be determinedin a temporal manner by repeated scans of interactions which occur ateach of a multiplicity of conditions. However, instead of testing eachindividual interaction separately, a multiplicity of sequenceinteractions may be simultaneously determined on a matrix.

A. Labeling Techniques

The target polynucleotide may be labeled by any of a number ofconvenient detectable markers. A fluorescent label is preferred becauseit provides a very strong signal with low background. It is alsooptically detectable at high resolution and sensitivity through a quickscanning procedure. Other potential labeling moieties include,radioisotopes, chemiluminescent compounds, labeled binding proteins,heavy metal atoms, spectroscopic markers, magnetic labels, and linkedenzymes.

Another method for labeling may bypass any label of the target sequence.The target may be exposed to the probes, and a double strand hybrid isformed at those positions only. Addition of a double strand specificreagent will detect where hybridization takes place. An intercalativedye such as ethidium bromide may be used as long as the probesthemselves do not fold back on themselves to a significant extentforming hairpin loops. See, e.g., Sheldon et al. (1986) U.S. Pat. No.4,582,789. However, the length of the hairpin loops in shortoligonucleotide probes would typically be insufficient to form a stableduplex.

In another embodiment, different targets may be simultaneously sequencedwhere each target has a different label. For instance, one target couldhave a green fluorescent label and a second target could have a redfluorescent label. The scanning step will distinguish sites of bindingof the red label from those binding the green fluorescent label. Eachsequence can be analyzed independently from one another.

Suitable chromogens will include molecules and compounds which absorblight in a distinctive range of wavelengths so that a color may beobserved, or emit light when irradiated with radiation of a particularwave length or wave length range, e.g., fluorescers. Biliproteins, e.g.,phycoerythrin, may also serve as labels.

A wide variety of suitable dyes are available, being primarily chosen toprovide an intense color with minimal absorption by their surroundings.Illustrative dye types include quinoline dyes, triarylmethane dyes,acridine dyes, alizarine dyes, phthaleins, insect dyes, azo dyes,anthraquinoid dyes, cyanine dyes, phenazathionium dyes, andphenazoxonium dyes.

A wide variety of fluorescers may be employed either by themselves or inconjunction with quencher molecules. Fluorescers of interest fall into avariety of categories having certain primary functionalities. Theseprimary functionalities include 1- and 2-aminonaphthalene,p,p'-diaminostilbenes, pyrenes, quaternary phenanthridine salts,9-aminoacridines, p,p'-diaminobenzophenone imines, anthracenes,oxacarbocyanine, merocyanine, 3-aminoequilenin, perylene,bis-benzoxazole, bis-p-oxazolyl benzene, 1,2-benzophenazin, retinol,bis-3-aminopyridinium salts, hellebrigenin, tetracycline, sterophenol,benzimidzaolylphenylamine, 2-oxo-3-chromen, indole, xanthen,7-hydroxycoumarin, phenoxazine, salicylate, strophanthidin, porphyrins,triarylmethanes and flavin. Individual fluorescent compounds which havefunctionalities for linking or which can be modified to incorporate suchfunctionalities include, e.g., dansyl chloride; fluoresceins such as3,6-dihydroxy-9-phenylxanthhydrol; rhodamineisothiocyanate; N-phenyl1-amino-8-sulfonatonaphthalene; N-phenyl 2-amino-6-sulfonatonaphthalene;4-acetamido-4-isothiocyanato-stilbene-2,2'-disulfonic acid;pyrene-3-sulfonic acid; 2-toluidinonaphthalene-6-sulfonate; N-phenyl,N-methyl 2-aminoaphthalene-6-sulfonate; ethidium bromide; stebrine;auromine-0,2-(9'-anthroyl)palmitate; dansyl phosphatidylethanolamine;N,N'-dioctadecyl oxacarbocyanine; N,N'-dihexyl oxacarbocyanine;merocyanine, 4-(3'pyrenyl)butyrate; d-3-aminodesoxy-equilenin;12-(9'-anthroyl)stearate; 2-methylanthracene; 9-vinylanthracene;2,2'-(vinylene-p-phenylene)bisbenzoxazole; p-bis2-(4-methyl-5-phenyl-oxazolyl)!benzene;6-dimethylamino-1,2-benzophenazin; retinol; bis(3'-aminopyridinium)1,10-decandiyl diiodide; sulfonaphthylhydrazone of hellibrienin;chlorotetracycline;N-(7-dimethylamino-4-methyl-2-oxo-3-chromenyl)maleimide; N-p-(2-benzimidazolyl)-phenyl!maleimide; N-(4-fluoranthyl)maleimide;bis(homovanillic acid); resazarin;4-chloro-7-nitro-2,1,3-benzooxadiazole; merocyanine 540; resorufin; rosebengal; and 2,4-diphenyl-3(2H)-furanone.

Desirably, fluorescers should absorb light above about 300 nm,preferably about 350 nm, and more preferably above about 400 nm, usuallyemitting at wavelengths greater than about 10 nm higher than thewavelength of the light absorbed. It should be noted that the absorptionand emission characteristics of the bound dye may differ from theunbound dye. Therefore, when referring to the various wavelength rangesand characteristics of the dyes, it is intended to indicate the dyes asemployed and not the dye which is unconjugated and characterized in anarbitrary solvent.

Fluorescers are generally preferred because by irradiating a fluorescerwith light, one can obtain a plurality of emissions. Thus, a singlelabel can provide for a plurality of measurable events.

Detectable signal may also be provided by chemiluminescent andbioluminescent sources. Chemiluminescent sources include a compoundwhich becomes electronically excited by a chemical reaction and may thenemit light which serves as the detectible signal or donates energy to afluorescent acceptor. A diverse number of families of compounds havebeen found to provide chemiluminescence under a variety of conditions.One family of compounds is 2,3-dihydro-1,-4-phthalazinedione. The mostpopular compound is luminol, which is the 5-amino compound. Othermembers of the family include the 5-amino-6,7,8-trimethoxy- and thedimethylamino ca!benz analog. These compounds can be made to luminescewith alkaline hydrogen peroxide or calcium hypochlorite and base.Another family of compounds is the 2,4,5-triphenylimidazoles, withlophine as the common name for the parent product. Chemiluminescentanalogs include para-dimethylamino and -methoxy substituents.Chemiluminescence may also be obtained with oxalates, usually oxalylactive esters, e.g., p-nitrophenyl and a peroxide, e.g., hydrogenperoxide, under basic conditions. Alternatively, luciferins may be usedin conjunction with luciferase or lucigenins to provide bioluminescence.

Spin labels are provided by reporter molecules with an unpaired electronspin which can be detected by electron spin resonance (ESR)spectroscopy. Exemplary spin labels include organic free radicals,transitional metal complexes, particularly vanadium, copper, iron, andmanganese, and the like. Exemplary spin labels include nitroxide freeradicals.

B. Scanning System

With the automated detection apparatus, the correlation of specificpositional labeling is converted to the presence on the target ofsequences for which the reagents have specificity of interaction. Thus,the positional information is directly converted to a databaseindicating what sequence interactions have occurred. For example, in anucleic acid hybridization application, the sequences which haveinteracted between the substrate matrix and the target molecule can bedirectly listed from the positional information. The detection systemused is described in Pirrung et al. (1992) U.S. Pat. No. 5,143,854; andSer. No. 07/624,120, now abandoned. Although the detection describedtherein is a fluorescence detector, the detector may be replaced by aspectroscopic or other detector. The scanning system may make use of amoving detector relative to a fixed substrate, a fixed detector with amoving substrate, or a combination. Alternatively, mirrors or otherapparatus can be used to transfer the signal directly to the detector.See, e.g, Ser. No. 07/624,120, now abandoned, which is herebyincorporated herein by reference.

The detection method will typically also incorporate some signalprocessing to determine whether the signal at a particular matrixposition is a true positive or may be a spurious signal. For example, asignal from a region which has actual positive signal may tend to spreadover and provide a positive signal in an adjacent region which actuallyshould not have one. This may occur, e.g., where the scanning system isnot properly discriminating with sufficiently high resolution in itspixel density to separate the two regions. Thus, the signal over thespatial region may be evaluated pixel by pixel to determine thelocations and the actual extent of positive signal. A true positivesignal should, in theory, show a uniform signal at each pixel location.Thus, processing by plotting number of pixels with actual signalintensity should have a clearly uniform signal intensity. Regions wherethe signal intensities show a fairly wide dispersion, may beparticularly suspect and the scanning system may be programmed to morecarefully scan those positions.

In another embodiment, as the sequence of a target is determined at aparticular location, the overlap for the sequence would necessarily havea known sequence. Thus, the system can compare the possibilities for thenext adjacent position and look at these in comparison with each other.Typically, only one of the possible adjacent sequences should give apositive signal and the system might be programmed to compare each ofthese possibilities and select that one which gives a strong positive.In this way, the system can also simultaneously provide some means ofmeasuring the reliability of the determination by indicating what theaverage signal to background ratio actually is.

More sophisticated signal processing techniques can be applied to theinitial determination of whether a positive signal exists or not. See,e.g., Ser. No. 07/624,120, now abandoned.

From a listing of those sequences which interact, data analysis may beperformed on a series of sequences. For example, in a nucleic acidsequence application, each of the sequences may be analyzed for theiroverlap regions and the original target sequence may be reconstructedfrom the collection of specific subsequences obtained therein. Othersorts of analyses for different applications may also be performed, andbecause the scanning system directly interfaces with a computer theinformation need not be transferred manually. This provides for theability to handle large amounts of data with very little humanintervention. This, of course, provides significant advantages overmanual manipulations. Increased throughput and reproducibility isthereby provided by the automation of a vast majority of steps in any ofthese applications.

XI. DATA ANALYSIS

A. General

Data analysis will typically involve aligning the proper sequences withtheir overlaps to determine the target sequence. Although the target"sequence" may not specifically correspond to any specific molecule,especially where the target sequence is broken and fragmented in thesequencing process, the sequence corresponds to a contiguous sequence ofthe subfragments.

The data analysis can be performed by a computer using an appropriateprogram. See, e.g., Drmanac, R. et al. (1989) Genomics 4:114-128; and acommercially available analysis program available from the GeneticEngineering Center, P.O. Box 794, 11000 Belgrade, Yugoslavia. Althoughthe specific manipulations necessary to reassemble the target sequencefrom fragments may take many forms, one embodiment uses a sortingprogram to sort all of the subsequences using a defined hierarchy. Thehierarchy need not necessarily correspond to any physical hierarchy, butprovides a means to determine, in order, which subfragments haveactually been found in the target sequence. In this manner, overlaps canbe checked and found directly rather than having to search throughoutthe entire set after each selection process. For example, where theoligonucleotide probes are 10-mers, the first 9 positions can be sorted.A particular subsequence can be selected as in the examples, todetermine where the process starts. As analogous to the theoreticalexample provided above, the sorting procedure provides the ability toimmediately find the position of the subsequence which contains thefirst 9 positions and can compare whether there exists more than 1subsequence during the first 9 positions. In fact, the computer caneasily generate all of the possible target sequences which contain givencombination of subsequences. Typically there will be only one, but invarious situations, there will be more.

An exemplary flow chart for a sequencing program is provided in FIG. 4.In general terms, the program provides for automated scanning of thesubstrate to determine the positions of probe and target interaction.Simple processing of the intensity of the signal may be incorporated tofilter out clearly spurious signals. The positions with positiveinteraction are correlated with the sequence specificity of specificmatrix positions, to generate the set of matching subsequences. Thisinformation is further correlated with other target sequenceinformation, e.g., restriction fragment analysis. The sequences are thenaligned using overlap data, thereby leading to possible correspondingtarget sequences which will, optimally, correspond to a single targetsequence.

B. Hardware

A variety of computer systems may be used to run a sequencing program.The program may be written to provide both the detecting and scanningsteps together and will typically be dedicated to a particular scanningapparatus. However, the components and functional steps may be separatedand the scanning system may provide an output, e.g., through tape or anelectronic connection into a separate computer which separately runs thesequencing analysis program. The computer may be any of a number ofmachines provided by standard computer manufacturers, e.g., IBMcompatible machines, Apple™ machines, VAX machines, and others, whichmay often use a UNIX™ operating system. Of course, the hardware used torun the analysis program will typically determine what programminglanguage would be used.

C. Software

Software would be easily developed by a person of ordinary skill in theprogramming art, following the flow chart provided, or based upon theinput provided and the desired result.

Of course, an exemplary embodiment is a polynucleotide sequence system.However, the theoretical and mathematical manipulations necessary fordata analysis of other linear molecules, such as polypeptides,carbohydrates, and various other polymers are conceptually similar.Simple branching polymers will usually also be sequencable using similartechnology. However, where there is branching, it may be desired thatadditional recognition reagents be used to determine the nature andlocation of branches. This can easily be provided by use of appropriatespecific reagents which would be generated by methods similar to thoseused to produce specific reagents for linear polymers.

XII. SUBSTRATE REUSE

Where a substrate is made with specific reagents that are relativelyinsensitive to the handling and processing steps involved in a singlecycle of use, the substrate may often be reused. The target moleculesare usually stripped off of the solid phase specific recognitionmolecules. Of course, it is preferred that the manipulations andconditions be selected as to be mild and to not affect the substrate.For example, if a substrate is acid labile, a neutral pH would bepreferred in all handling steps. Similar sensitivities would becarefully respected where recycling is desired.

A. Removal of Label

Typically for a recycling, the previously attached specific interactionwould be disrupted and removed. This will typically involve exposing thesubstrate to conditions under which the interaction between probe andtarget is disrupted. Alternatively, it may be exposed to conditionswhere the target is destroyed. For example, where the probes areoligonucleotides and the target is a polynucleotide, a heating and lowsalt wash will often be sufficient to disrupt the interactions.Additional reagents may be added such as detergents, and organic orinorganic solvents which disrupt the interaction between the specificreagents and target. In an embodiment where the specific reagents areantibodies, the substrate may be exposed to a gentle detergent whichwill denature the specific binding between the antibody and its target.The conditions are selected to avoid severe disruption or destruction ofthe structure of the antibody and to maintain the specificity of theantibody binding site. Conditions with specific pH, detergentconcentration, salt concentration, ionic concentration, and otherparameters may be selected which disrupt the specific interactions.

B. Storage and Preservation

As indicated above, the matrix will typically be maintained underconditions where the matrix itself and the linkages and specificreagents are preserved. Various specific preservatives may be addedwhich prevent degradation. For example, if the reagents are acid or baselabile, a neutral pH buffer will typically be added. It is also desiredto avoid destruction of the matrix by growth of organisms which maydestroy organic reagents attached thereto. For this reason, apreservative such as cyanide or azide may be added. However, thechemical preservative should also be selected to preserve the chemicalnature of the linkages and other components of the substrate. Typically,a detergent may also be included.

C. Processes to Avoid Degradation of Oligomers

In particular, a substrate comprising a large number of oligomers willbe treated in a fashion which is known to maintain the quality andintegrity of oligonucleotides. These include storing the substrate in acarefully controlled environment under conditions of lower temperature,cation depletion (EDTA and EGTA), sterile conditions, and inert argon ornitrogen atmosphere.

XIII. INTEGRATED SEQUENCING STRATEGY

A. Initial Mapping Strategy

As indicated above, although the VLSIPS™ technology may be applied tosequencing embodiments, it is often useful to integrate other conceptsto simplify the sequencing. For example, nucleic acids may be easilysequenced by careful selection of the vectors and hosts used foramplifying and generating the specific target sequences. For example, itmay be desired to use specific vectors which have been designed tointeract most efficiently with the VLSIPS substrate. This is alsoimportant in fingerprinting and mapping strategies. For example, vectorsmay be carefully selected having particular complementary sequenceswhich are designed to attach to a genetic or specific oligomer on thesubstrate. This is also applicable to situations where it is desired totarget particular sequences to specific locations on the matrix.

In one embodiment, unnatural oligomers may be used to target naturalprobes to specific locations on the VLSIPS substrate. In addition,particular probes may be generated for the mapping embodiment which aredesigned to have specific combinations of characteristics. For example,the construction of a mapping substrate may depend upon use of anotherautomated apparatus which takes clones isolated from a chromosome walkand attaches them individually or in bulk to the VLSIPS substrate.

In another embodiment, a variety of specific vectors having known andparticular "targeting" sequences adjacent to the cloning sites may beindividually used to clone a selected probe, and the isolated probe willthen be targetable to a site on the VLSIPS substrate with a sequencecomplementary to the "target" sequence.

B. Selection of Smaller Clones

In the fingerprinting and mapping embodiments, the selection of probesmay be very important. Significant mathematical analysis may be appliedto determine which specific sequences should be used as those probes. Ofcourse, for fingerprinting use, these sequences would be most desiredthat show significant heterogeneity across the human population.Selection of the specific sequences which would most favorably beutilized will tend to be single copy sequences within the genome.

Various hybridization selection procedures may be applied to selectsequences which tend not to be repeated within a genome, and thus wouldtend to be conserved across individuals. For example, hybridizationselections may be made for non-repetitive and single copy sequences.See, e.g., Britten and Kohne (1968) "Repeated Sequences in DNA," Science161:529-540. On the other hand, it may be desired under certaincircumstances to use repeated sequences. For example, where afingerprint may be used to identify or distinguish different species, orwhere repetitive sequences may be diagnostic of specific species,repetitive sequences may be desired for inclusion in the fingerprintingprobes. In either case, the sequencing capability will greatly assist inthe selection of appropriate sequences to be used as probes.

Also as indicated above, various means for constructing an appropriatesubstrate may involve either mechanical or automated procedures. Thestandard VLSIPS automated procedure involves synthesizingoligonucleotides or short polymers directly on the substrate. In variousother embodiments, it is possible to attach separately synthesizedreagents onto the matrix in an ordered array. Other circumstances maylend themselves to transfer a pattern from a petri plate onto a solidsubstrate. Also, there are methods for site specifically directingcollections of reagents to specific locations using unnaturalnucleotides or equivalent sorts of targeting molecules.

While a brute force manual transfer process may be utilized sequentiallyfor attaching various samples to successive positions, instrumentationfor automating such procedures may also be devised. The automated systemfor performing such would preferably be relatively easily designed andconceptually easily understood.

XIV. COMMERCIAL APPLICATIONS

A. Sequencing

As indicated above, sequencing may be performed either de novo or as averification of another sequencing method. The present hybridizationtechnology provides the ability to sequence nucleic acids andpolynucleotides de novo, or as a means to verify either the Maxam andGilbert chemical sequencing technique or Sanger and Coulson dideoxy-sequencing techniques. The hybridization method is useful to verifysequencing determined by any other sequencing technique and to closelycompare two similar sequences, e.g., to identify and locate sequencedifferences.

Besides polynucleotide sequencing, the present invention also providesmeans for sequencing other polymers. This includes polypeptides,carbohydrates, synthetic organic polymers, and other polymers. Again,the sequencing may be either verification or de novo.

Of course, sequencing can be very important in many different sorts ofenvironments. For example, it will be useful in determining the geneticsequence of particular markers in various individuals. In addition,polymers may be used as markers or for information containing moleculesto encode information. For example, a short polynucleotide sequence maybe included in large bulk production samples indicating themanufacturer, date, and location of manufacture of a product. Forexample, various drugs may be encoded with this information with a smallnumber of molecules in a batch. For example, a pill may have somewherefrom 10 to 100 to 1,000 or more very short and small molecules encodingthis information. When necessary, this information may be decoded from asample of the material using a polymerase chain reaction (PCR) or otheramplification method. This encoding system may be used to provide theorigin of large bulky samples without significantly affecting theproperties of those samples. For example, chemical samples may also beencoded by this method thereby providing means for identifying thesource and manufacturing details of lots. The origin of bulk hydrocarbonsamples may be encoded. Production lots of organic compounds such asbenzene or plastics may be encoded with a short molecule polymer. Foodstuffs may also be encoded using similar marking molecules. Even toxicwaste samples can be encoded determining the source or origin. In thisway, proper disposal can be traced or more easily enforced.

Similar sorts of encoding may be provided by fingerprinting-typeanalysis. Whether the resolution is absolute or less so, the concept ofcoding information on molecules such as nucleic acids, which can beamplified and later decoded, may be a very useful and importantapplication.

This technology also provides the ability to include markers for originsof biological materials. For example, a patented animal line may betransformed with a particular unnatural sequence which can be tracedback to its origin. With a selection of multiple markers, the likelihoodcould be negligible that a combination of markers would haveindependently arisen from a source other than the patented orspecifically protected source. This technique may provide a means fortracing the actual origin of particular biological materials. Bacteria,plants, and animals will be subject to marking by such encodingsequences.

B. Fingerprinting

As indicated above, fingerprinting technology may also be used for dataencryption. Moreover, fingerprinting allows for significantidentification of particular individuals. Where the fingerprintingtechnology is standardized, and used for identification of large numbersof people, related equipment and peripheral processing will be developedto accompany the underlying technology. For example, specific equipmentmay be developed for automatically taking a biological sample andgenerating or amplifying the information molecules within the sample tobe used in fingerprinting analysis. Moreover, the fingerprintingsubstrate may be mass produced using particular types of automaticequipment. Synthetic equipment may produce the entire matrixsimultaneously by stepwise synthetic methods as provided by the VLSIPS™technology. The attachment of specific probes onto a substrate may alsobe automated, e.g., making use of the caged biotin technology. See,e.g., Barrett et al. (1993) U.S. Pat. No. 5,252,743. As indicated above,there are automated methods for actually generating the matrix andsubstrate with distinct sequence reagents positionally located at eachof the matrix positions. Where such reagents are, e.g., unnatural aminoacids, a targeting function may be utilized which does not interferewith a natural nucleotide functionality.

In addition, peripheral processing may be important and may be dedicatedto this specific application. Thus, automated equipment for producingthe substrates may be designed, or particular systems which take in abiological sample and output either a computer readout or an encodedinstrument, e.g., a card or document which indicates the information andcan provide that information to others. An identification having a shortmagnetic strip with a few million bits may be used to provide individualidentification and important medical information useful in a medicalemergency.

In fact, data banks may be set up to correlate all of this informationof fingerprinting with medical information. This may allow for thedetermination of correlations between various medical problems andspecific DNA sequences. By collating large populations of medicalrecords with genetic information, genetic propensities and geneticsusceptibilities to particular medical conditions may be developed.Moreover, with standardization of substrates, the micro encoding datamay be also standardized to reproduce the information from a centralizeddata bank or on an encoding device carried on an individual person. Onthe other hand, if the fingerprinting procedure is sufficiently quickand routine, every hospital may routinely perform a fingerprintingoperation and from that determine many important medical parameters foran individual.

In particular industries, the VLSIPS sequencing, fingerprinting, ormapping technology will be particularly appropriate. As mentioned above,agricultural livestock suppliers may be able to encode and determinewhether their particular strains are being used by others. Byincorporating particular markers into their genetic stocks, the markerswill indicate origin of genetic material. This is applicable to seedproducers, livestock producers, and other suppliers of medical oragricultural biological materials.

This may also be useful in identifying individual animals or plants. Forexample, these markers may be useful in determining whether certain fishreturn to their original breeding grounds, whether sea turtles alwaysreturn to their original birthplaces, or to determine the migrationpatterns and viability of populations of particular endangered species.It would also provide means for tracking the sources of particularanimal products. For example, it might be useful for determining theorigins of controlled animal substances such as elephant ivory orparticular bird populations whose importation or exportation iscontrolled.

As indicated above, polymers may be used to encode important informationon source and batch and supplier. This is described in greater detail,e.g., "Applications of PCR to industrial problems," (1990) in Chemicaland Engineering News 68:145, which is hereby incorporated herein byreference. In fact, the synthetic method can be applied to the storageof enormous amounts of information. Small substrates may encode enormousamounts of information, and its recovery will make use of the inherentreplication capacity. For example, on regions of 10 μm×10 μm, 1 cm² has10⁶ regions. In theory, the entire human genome could be attached in1000 nucleotide segments on a 3 cm² surface. Genomes of endangeredspecies may be stored on these substrates.

Fingerprinting may also be used for genetic tracing or for identifyingindividuals for forensic science purposes. See, e.g., Morris, J. et al.(1989) "Biostatistical Evaluation of Evidence From Continuous AlleleFrequency Distribution DNA Probes in Reference to Disputed Paternity andIdentity," J. Forensic Science 34:1311-1317, and references providedtherein; each of which is hereby incorporated herein by reference.

In addition, the high resolution fingerprinting allows thedistinguishability to high resolution of particular samples. Asindicated above, new cell classifications may be defined based oncombinations of a large number of properties. Similar applications willbe found in distinguishing different species of animals or plants. Infact, microbial identification may become dependent on characterizationof the genetic content. Tumors or other cells exhibiting abnormalphysiology will be detectable by use of the present invention. Also,knowing the genetic fingerprint of a microorganism may provide veryuseful information on how to treat an infection by such organism.

Modifications of the fingerprint embodiments may be used to diagnose thecondition of the organism. For example, a blood sample is presently usedfor diagnosing any of a number of different physiological conditions. Amulti-dimensional fingerprinting method made available by the presentinvention could become a routine means for diagnosing an enormous numberof physiological features simultaneously. This may revolutionize thepractice of medicine in providing information on an enormous number ofparameters together at one time. In another way, the geneticpredisposition may also revolutionize the practice of medicine providinga physician with the ability to predict the likelihood of particularmedical conditions arising at any particular moment. It also providesthe ability to apply preventive medicine.

The present invention might also find application in use for screeningnew drugs and new reagents which may be very important in medicaldiagnosis or other applications. For example, a description ofgenerating a population of monoclonal antibodies with definedspecificities may be very useful for producing various drugs ordiagnostic reagents.

Also available are kits with the reagents useful for performingsequencing, fingerprinting, and mapping procedures. The kits will havevarious compartments with the desired necessary reagents, e.g.,substrate, labeling reagents for target samples, buffers, and otheruseful accompanying products.

C. Mapping

The present invention also provides the means for mapping sequenceswithin enormous stretches of sequence. For example, nucleotide sequencesmay be mapped within enormous chromosome size sequence maps. Forexample, it would be possible to map a chromosomal location within thechromosome which contains hundreds of millions of nucleotide base pairs.In addition, the mapping and fingerprinting embodiments allow fortesting of chromosomal translocations, one of the standard problems forwhich amniocentesis is performed.

Thus, the present invention provides a powerful tool and the means forperforming sequencing, fingerprinting, and mapping functions onpolymers. Although most easily and directly applicable topolynucleotides, polypeptides, carbohydrates, and other sorts ofmolecules can be advantageously utilized using the present technology.

The present invention will be better understood by reference to thefollowing illustrative examples. The following examples are offered byway of illustration and not by way of limitation.

EXPERIMENTAL

I. Sequencing

A. polynucleotide

B. polypeptide

C. short peptide

1. Herz antibody identification

II. Fingerprinting

A. polynucleotide fingerprint

B. peptide fingerprint

C. cell classification scheme

D. temporal development scheme

1. developmental antigens

2. developmental mRNA expression

E. diagnostic test

1. viral identification

2. bacterial identification

3. other microbiological identifications

4. allergy test (immobilized antigens)

F. individual (animal/plant) identification

1. genetic

2. immunological

G. genetic screen

1. test alleles with markers

2. amniocentesis

III. Mapping

A. positionally located clones (caged biotin)

1. short probes, long targets

2. long targets, short probes

B. positionally defined clones

IV. Conclusion

Relevant applications whose techniques are incorporated herein byreference are Pirrung, et al., Ser. No. 07/362,901, filed Jun. 7, 1989,now abandoned; Pirrung et al. (1992) U.S. Pat. No. 5,143,854; Barrett,et al., Ser. No. 07/435,316 filed Nov. 13, 1989, now abandoned; Barrett,et al. (1993) U.S. Pat. No. 5,252,743; and commonly assigned andsimultaneously filed applications Ser. No. 07/624,120, now abandoned,and Ser. No. 07/626,730.

Also, additional relevant techniques are described, e.g., in Sambrook,J., et al. (1989) Molecular Cloning: a Laboratory Manual, 2d Ed., vols1-3, Cold Spring Harbor Press, New York; Greenstein and Winitz (1961)Chemistry of the Amino Acids, Wiley and Sons, New York; Bodzansky, M.(1988) Peptide Chemistry: a Practical Textbook, Springer-Verlag, NewYork; Harlow and Lane (1988) Antibodies: A Laboratory Manual, ColdSpring Harbor Press, New York; Glover, D. (ed.) (1987) DNA Cloning: APractical Approach, vols 1-3, IRL Press, Oxford; Bishop and Rawlings(1987) Nucleic Acid and Protein Sequence Analysis: A Practical Approach,IRL Press, Oxford; Hames and Higgins (1985) Nucleic Acid Hybridisation:A Practical Approach, IRL Press, Oxford; Wu et al. (1989) RecombinantDNA Methodology, Academic Press, San Diego; Goding (1986) MonoclonalAntibodies: Principles and Practice, (2d ed.), Academic Press, SanDiego; Finegold and Barron (1986) Bailey and Scott's DiagnosticMicrobiology, (7th ed.), Mosby Co., St. Louis; Collins et al. (1989)Microbiological Methods, (6th ed.), Butterworth, London; Chaplin andKennedy (1986) Carbohydrate Analysis: A Practical Approach, IRL Press,Oxford; Van Dyke (ed.) (1985) Bioluminescence and Chemiluminescence:Instruments and Applications, vol 1, CRC Press, Boca Rotan; and Ausubelet al. (ed.) (1990) Current Protocols in Molecular Biology, GreenePublishing and Wiley-Interscience, New York; each of which is herebyincorporated herein by reference.

The following examples are provided to illustrate the efficacy of theinventions herein. All operations were conducted at about ambienttemperatures and pressures unless indicated to the contrary.

I. SEQUENCING

A. Polynucleotide

1. HPLC of the photolysis of 5'-O-nitroveratryl-thymidine.

In order to determine the time for photolysis of 5'-nitroveratrylthymidine to thymidine a 100 μM solution of NV-Thym-OH(5'-O-nitroveratryl thymidine) in dioxane was made and .sup.˜ 200 μlaliquots were irradiated (in a quartz cuvette 1 cm×2 mm) at 362.3 nm for20 sec, 40 sec, 60 sec, 2 min, 5 min, 10 min, 15 min, and 20 min. Theresulting irradiated mixtures were then analyzed by HPLC using a VarianMicroPak SP column (C₁₈ analytical) at a flow rate of 1 ml/min and asolvent system of 40% CH₃ CN and 60% water. Thymidine has a retentiontime of 1.2 min and NVO-Thym-OH has a retention time of 2.1 min. It wasseen that after 10 min of exposure the deprotection was complete.

2. Preparation and Detection of Thymidine-Cytidine dimer (FITC)

The reaction is illustrated: ##STR15##

To an aminopropylated glass slide (standard VLSIPS™ Technology) wasadded a mixture of the following:

12.2 mg of NVO-Thym-CO₂ H (IX)

3.4 mg of HOBT (N-hydroxybenztriazal)

8.8 μl DIEA (Diisopropylethylamine)

11.1 mg BOP reagent

2.5 ml DMF

After 2 h coupling time (standard VLSIPS) the plate was washed,acetylated with acetic anhydride/pyridine, washed, dried, and photolyzedin dioxane at 362 nm at 14 mW/cm² for 10 min using a 500 μm checkerboardmask. The slide was then taken and treated with a mixture of thefollowing:

107 mg of FMOC-amine modified C (III)

21 mg of tetrazole

1 ml anhydrous CH₃ CN

After being treated for approximately 8 min, the slide was washed offwith CH₃ CN, dried, and oxidized with I₂ /H₂ O/THF/lutidine for 1 min.The slide was again washed, dried, and treated for 30 min with a 20%solution of DBU in DMF. After thorough rinsing of the slide, it was nextexposed to a FITC solution (1 mM fluorescein isothiocyanate FITC! inDMF) for 50 min, then washed, dried, and examined by fluorescencemicroscopy. This reaction is illustrated: ##STR16## 3. Preparation andDetection of Thymidine-Cytidine dimer (Biotin)

An aminopropyl glass slide, was soaked in a solution of ethylene oxide(20% in DMF) to generate a hydroxylated surface. The slide was added toa mixture of the following:

32 mg of NVO-T-OCED (X)

11 mg of tetrazole

0.5 ml of anhydrous CH₃ CN

After 8 min the plate was then rinsed with acetonitrile, then oxidizedwith I₂ /H₂ O/THF/lutidine for 1 min, washed and dried. The slide wasthen exposed to a 1:3 mixture of acetic anhydride:pyridine for 1 h, thenwashed and dried. The substrate was then photolyzed in dioxane at 362 nmat 14 mW/cm² for 10 min using a 500 μm checkerboard mask, dried, andthen treated with a mixture of the following:

65 mg of biotin modified C (IV)

11 mg of tetrazole

0.5 ml anhydrous CH₃ CN

After 8 min the slide was washed with CH₃ CN then oxidized with I₂ /H₂O/THF/lutidine for 1 min, washed, and then dried. The slide was thensoaked for 30 min in a PBS/0.05% Tween 20 buffer and the solution thenshaken off. The slide was next treated with FITC-labeled streptavidin at10 μg/ml in the same buffer system for 30 min. After this time thestreptavidin-buffer system was rinsed off with fresh PBS/0.05% Tween 20buffer and then the slide was finally agitated in distilled water forabout 1/2 h. After drying, the slide was examined by fluorescencemicroscopy (see FIG. 2 and FIG. 3).

4. substrate preparation

Before attachment of reactive groups it is preferred to clean thesubstrate which is, in a preferred embodiment, a glass substrate such asa microscope slide or cover slip. A roughened surface will be useablebut a plastic or other solid substrate is also appropriate. According toone embodiment the slide is soaked in an alkaline bath consisting of,e.g., 1 liter of 95% ethanol with 120 ml of water and 120 grams ofsodium hydroxide for 12 hours. The slides are washed with a buffer andunder running water, allowed to air dry, and rinsed with a solution of95% ethanol.

The slides are then aminated with, e.g., aminopropyltriethoxysilane forthe purpose of attaching amino groups to the glass surface on linkermolecules, although other omega functionalized silanes could also beused for this purpose. In one embodiment 0.1% aminopropyltriethoxysilaneis utilized, although solutions with concentrations from 10⁻⁷ % to 10%may be used, with about 10⁻³ % to 2% preferred. A 0.1% mixture isprepared by adding to 100 ml of a 95% ethanol/5% water mixture, 100microliters (μl) of aminopropyltriethoxysilane. The mixture is agitatedat about ambient temperature on a rotary shaker for an appropriateamount of time, e.g., about 5 minutes. 500 μl of this mixture is thenapplied to the surface of one side of each cleaned slide. After 4minutes or more, the slides are decanted of this solution and thoroughlyrinsed three times or more by dipping in 100% ethanol.

After the slides dry, they are heated in a 110-120° C. vacuum oven forabout 20 minutes, and then allowed to cure at room temperature for about12 hours in an argon environment. The slides are then dipped into DMF(dimethylformamide) solution, followed by a thorough washing withmethylene chloride.

5. linker attachment, blocking of free sites

The aminated surface of the slide is then exposed to about 500 μl of,for example, a 30 millimolar (mM) solution of NVOC-nucleotide- NHS(N-hydroxysuccinimide) in DMF for attachment of a NVOC-nucleotide toeach of the amino groups. See, e.g., SIGMA Chemical Company for variousnucleotide derivatives. The surface is washed with, for example, DMF,methylene chloride, and ethanol.

Any unreacted aminopropyl silane on the surface, i.e., those aminogroups which have not had the NVOC-nucleotide attached, are now cappedwith acetyl groups (to prevent further reaction) by exposure to a 1:3mixture of acetic anhydride in pyridine for 1 hour. Other materialswhich may perform this residual capping function include trifluoroaceticanhydride, formicacetic anhydride, or other reactive acylating agents.Finally, the slides are washed again with DMF, methylene chloride, andethanol.

6. synthesis of eight trimers of C and T

FIG. 4 illustrates a possible synthesis of the eight trimers of thetwo-monomer set: cytosine and thymine (represented by C and T,respectively). A glass slide bearing silane groups terminating in6-nitroveratryloxycarboxamide (NVOC-NH) residues is prepared as asubstrate. Active esters (pentafluorophenyl, OBt, etc.) of cytosine andthymine protected at the 5' hydroxyl group with NVOC are prepared asreagents. While not pertinent to this example, if side chain protectinggroups are required for the monomer set, these must not be photoreactiveat the wavelength of light used to protect the primary chain.

For a monomer set of size n, n×l cycles are required to synthesize allpossible sequences of length l. A cycle consists of:

1. Irradiation through an appropriate mask to expose the 5'--OH groupsat the sites where the next residue is to be added, with appropriatewashes to remove the by-products of the deprotection.

2. Addition of a single activated and protected (with the samephotochemically-removable group) monomer, which will react only at thesites addressed in step 1, with appropriate washes to remove the excessreagent from the surface.

The above cycle is repeated for each member of the monomer set untileach location on the surface has been extended by one residue in oneembodiment. In other embodiments, several residues are sequentiallyadded at one location before moving on to the next location. Cycle timeswill generally be limited by the coupling reaction rate, now as short asabout 10 min in automated oligonucleotide synthesizers. This step isoptionally followed by addition of a protecting group to stabilize thearray for later testing. For some types of polymers (e.g., peptides), afinal deprotection of the entire surface (removal of photoprotectiveside chain groups) may be required.

More particularly, as shown in FIG. 4A, the glass 20 is provided withregions 22, 24, 26, 28, 30, 32, 34, and 36. Regions 30, 32, 34, and 36are masked, indicated by the hatched regions, as shown in FIG. 4B andthe glass is irradiated by the bright regions 22, 24, 26, and 28, andexposed to a reagent containing a photosensitive blocked C (e.g.,cytosine derivative), with the resulting structure shown in FIG. 4C. Thesubstrate is carefully washed and the reactants removed. Thereafter,regions 22, 24, 26, and 28 are masked, as indicated by the hatchedregion, the glass is irradiated (as shown in FIG. 4D), as indicated bythe bright regions, at 30, 32, 34, and 36, and exposed to aphotosensitive blocked reagent containing T (e.g., thymine derivative),with the resulting structure shown in FIG. 4E. The process proceeds,consecutively masking and exposing the sections as shown until thestructure shown in FIG. 4M is obtained. The glass is irradiated and theterminal groups are, optionally, capped by acetylation. As shown, allpossible trimers of cytosine/thymine are obtained.

In this example, no side chain protective group removal is necessary, asmight be common in modified nucleotides. If it is desired, side chaindeprotection may be accomplished by treatment with ethanedithiol andtrifluoroacetic acid.

In general, the number of steps needed to obtain a particular polymerchain is defined by:

    n×l                                                  (1)

where:

n=the number of monomers in the basis set of monomers, and

l=the number of monomer units in a polymer chain.

Conversely, the synthesized number of sequences of length l will be:

    n.sup.l.                                                   (2)

Of course, greater diversity is obtained by using masking strategieswhich will also include the synthesis of polymers having a length ofless than l. If, in the extreme case, all polymers having a length lessthan or equal to l are synthesized, the number of polymers synthesizedwill be:

    n.sup.l +n.sup.l-1 + . . . +n.sup.1.                       (3)

The maximum number of lithographic steps needed will generally be n foreach "layer" of monomers, i.e., the total number of masks (and,therefore, the number of lithographic steps) needed will be n×l. Thesize of the transparent mask regions will vary in accordance with thearea of the substrate available for synthesis and the number ofsequences to be formed. In general, the size of the synthesis areas willbe:

size of synthesis areas=(A)/(S)

where:

A is the total area available for synthesis; and

S is the number of sequences desired in the area.

It will be appreciated by those of skill in the art that the abovemethod could readily be used to simultaneously produce thousands ormillions of oligomers on a substrate using the photolithographictechniques disclosed herein. Consequently, the method results in theability to practically test large numbers of, for example, di, tri,tetra, penta, hexa, hepta, octa, nona, deca, even dodecanucleotides, orlarger polynucleotides (or correspondingly, polypeptides).

The above example has illustrated the method by way of a manual example.It will of course be appreciated that automated or semi-automatedmethods could be used. The substrate would be mounted in a flow cell forautomated addition and removal of reagents, to minimize the volume ofreagents needed, and to more carefully control reaction conditions.Successive masks will be applicable manually or automatically. See,e.g., Pirrung et al. (1992) U.S. Pat. No. 5,143,854 and Ser. No.07/624,120, now abandoned.

7. labeling of target

The target oligonucleotide can be labeled using standard proceduresreferred to above. As discussed, for certain situations, a reagent whichrecognizes interaction, e.g., ethidium bromide, may be provided in thedetection step. Alternatively, fluorescence labeling techniques may beapplied, see, e.g., Smith, et al. (1986) Nature, 321: 674-679; andProber, et al. (1987) Science, 238:336-341. The techniques describedtherein will be followed with minimal modifications as appropriate forthe label selected.

8. dimers of A, C, G, and T

The described technique may be applied, with photosensitive blockednucleotides corresponding to adenine, cytosine, guanine, and thymine, tomake combinations of polynucleotides consisting of each of the fourdifferent nucleotides. All 16 possible dimers would be made using aminor modification of the described method.

9. 10-mers of A, C, G, and T

The described technique for making dimers of A, C, G, and T may befurther extended to make longer oligonucleotides. The automated systemdescribed, e.g., in Pirrung et al. (1992) U.S. Pat. No. 5,143,854, andSer. No. 07/624,120, now abandoned, can be adapted to make all possible10-mers composed of the 4 nucleotides A, C, G, and T. Thephotosensitive, blocked nucleotide analogues have been described above,and would be readily adaptable to longer oligonucleotides.

10. specific recognition hybridization to 10-mers

The described hybridization conditions are directly applicable to thesequence specific recognition reagents attached to the substrate,produced as described immediately above. The 10-mers have an inherentproperty of hybridizing to a complementary sequence. For optimumdiscrimination between full matching and some mismatch, the conditionsof hybridization should be carefully selected, as described above.Careful control of the conditions, and titration of parameters should beperformed to determine the optimum collective conditions.

11. hybridization

Hybridization conditions are described in detail, e.g., in Hames andHiggins (1985) Nucleic Acid Hybridisation: A Practical Approach; and theconsiderations for selecting particular conditions are described, e.g.,in Wetmur and Davidson, (1988) J. Mol. Biol. 31:349-370, and Wood et al.(1985) Proc. Natl. Acad. Sci. USA 82:1585-1588. As described above,conditions are desired which can distinguish matching along the entirelength of the probe from where there is one or more mismatched bases.The length of incubation and conditions will be similar, in manyrespects, to the hybridization conditions used in Southern blottransfers. Typically, the GC bias may be minimized by the introductionof appropriate concentrations of the alkylammonium buffers, as describedabove.

Titration of the temperature and other parameters is desired todetermine the optimum conditions for specificity and distinguishabilityof absolutely matched hybridization from mismatched hybridization.

A fluorescently labeled target or set of targets are generated, asdescribed in Prober, et al. (1987) Science 238:336-341, or Smith, et al.(1986) Nature 321:674-679. Preferably, the target or targets are of thesame length as, or slightly longer, than the oligonucleotide probesattached to the substrate and they will have known sequences. Thus, onlya few of the probes hybridize perfectly with the target, and whichparticular ones did would be known.

The substrate and probes are incubated under appropriate conditions fora sufficient period of time to allow hybridization to completion. Thetime is measured to determine when the probe-target hybridizations havereached completion. A salt buffer which minimizes GC bias is preferred,incorporating, e.g., buffer, such as tetramethyl ammonium or tetraethylammonium ion at between about 2.4 and 3.0 M. See Wood, et al. (1985)Proc. Nat'l Acad. Sci. USA 82:1585-1588. This time is typically at leastabout 30 min, and may be as long as about 1-5 days. Typically very longmatches will hybridize more quickly, very short matches will hybridizeless quickly, depending upon relative target and probe concentrations.The hybridization will be performed under conditions where the reagentsare stable for that time duration.

Upon maximal hybridization, the conditions for washing are titrated.Three parameters initially titrated are time, temperature, and cationconcentration of the wash step. The matrix is scanned at various timesto determine the conditions at which the distinguishability between trueperfect hybrid and mismatched hybrid is optimized. These conditions willbe preferred in the sequencing embodiments.

12. positional detection of specific interaction

As indicated above, the detection of specific interactions may beperformed by detecting the positions where the labeled target sequencesare attached. Where the label is a fluorescent label, the apparatusdescribed, e.g., in Pirrung et al. (1992) U.S. Pat. No. 5,143,854; andSer. No. 07/624,120, now abandoned, may be advantageously applied. Inparticular, the synthetic processes described above will result in amatrix pattern of specific sequences attached to the substrate, and aknown pattern of interactions can be converted to correspondingsequences.

In an alternative embodiment, a separate reagent which differentiallyinteracts with the probe and interacted probe/targets can indicate whereinteraction occurs or does not occur. A single-strand specific reagentwill indicate where no interaction has taken place, while adouble-strand specific reagent will indicate where interaction has takenplace. An intercalating dye, e.g., ethidium bromide, may be used toindicate the positions of specific interaction.

13. analysis

Conversion of the positional data into sequence specificity will providethe set of subsequences whose analysis by overlap segments, may beperformed, as described above. Analysis is provided by the methodologydescribed above, or using, e.g., software available from the GeneticEngineering Center, P.O. Box 794, 11000 Belgrade, Yugoslavia (Yugoslavgroup). See, also, Macevicz, PCT publication no. WO 90/04652, which ishereby incorporated herein by reference.

B. Polypeptide

The description of the preparation of short peptides on a substrateincorporates by reference sections in Pirrung et al. (1992) U.S. Pat.No. 5,143,854, and described below.

1. slide preparation

Preparation of the substrate follows that described above fornucleotides.

2. linker attachment, blocking of free sites

The aminated surface of the slide is exposed to about 500 μl of, e.g., a30 millimolar (mM) solution of NVOC-GABA (gamma amino butyric acid) NHS(N-hydroxysuccinimide) in DMF for attachment of a NVOC-GABA to each ofthe amino groups. The surface is washed with, for example, DMF,methylene chloride, and ethanol. See Ser. No. 07,624,120, now abandoned,for details on amino acid chemistry.

Any unreacted aminopropyl silane on the surface, i.e., those aminogroups which have not had the NVOC-GABA attached, are now capped withacetyl groups (to prevent further reaction) by exposure to a 1:3 mixtureof acetic anhydride in pyridine for 1 hour. Other materials which mayperform this residual capping function include trifluoroaceticanhydride, formicacetic anhydride, or other reactive acylating agents.Finally, the slides are washed again with DMF, methylene chloride, andethanol.

3. synthesis of 8 trimers of "A" and "B"

See Pirrung et al. (1992) U.S. Pat. No. 5,143,854 which describes thepreparation of glycine and phenylalanine trimers. The technique issimilar to the method described above for making triners of C and T, butsubstituting photosensitive blocked glycine for the C derivative andphotosensitive blocked phenylalamine for the T derivative.

4. synthesis of a dimer of an aminopropyl group and a fluorescent group

In synthesizing the dimer of an aminopropyl group and a fluorescentgroup, a functionalized Durapore™ membrane was used as a substrate. TheDurapore™ membrane was a polyvinylidine difluoride with aminopropylgroups. The aminopropyl groups were protected with the DDZ group byreaction of the carbonyl chloride with the amino groups, a reactionreadily known to those of skill in the art. The surface bearing thesegroups was placed in a solution of THF and contacted with a mask bearinga checkerboard pattern of 1 mm opaque and transparent regions. The maskwas exposed to ultraviolet light having a wavelength down to at leastabout 280 nm for about 5 minutes at ambient temperature, although a widerange of exposure times and temperatures may be appropriate in variousembodiments of the invention. For example, in one embodiment, anexposure time of between about 1 and 5000 seconds may be used at processtemperatures of between -70 and +50° C.

In one preferred embodiment, exposure times of between about 1 and 500seconds at about ambient pressure are used. In some preferredembodiments, pressure above ambient is used to prevent evaporation.

The surface of the membrane was then washed for about 1 hour with afluorescent label which included an active ester bound to a chelate of alanthanide. Wash times will vary over a wide range of values from abouta few minutes to a few hours. These materials fluoresce in the red andthe green visible region. After the reaction with the active ester inthe fluorophore was complete, the locations in which the fluorophore wasbound could be visualized by exposing them to ultraviolet light andobserving the red and the green fluorescence. It was observed that thederivatized regions of the substrate closely corresponded to theoriginal pattern of the mask.

5. demonstration of signal capability

Signal detection capability was demonstrated using a low-level standardfluorescent bead kit manufactured by Flow Cytometry Standards and havingmodel no. 824. This kit includes 5.8 μm diameter beads, each impregnatedwith a known number of fluorescein molecules.

One of the beads was placed in the illumination field on the scan stagein a field of a laser spot which was initially shuttered. After beingpositioned in the illumination field, the photon detection equipment wasturned on. The laser beam was unblocked and it interacted with theparticle bead, which then fluoresced. Fluorescence curves of beadsimpregnated with 7,000 and 29,000 fluorescein molecules, are shown inFIGS. 11A and 11B, respectively of Pirrung et al. (1992) U.S. Pat. No.5,143,854. On each curve, traces for beads without fluorescein moleculesare also shown. These experiments were performed with 488 nm excitation,with 100 μW of laser power. The light was focused through a 40 power0.75 NA objective.

The fluorescence intensity in all cases started off at a high value andthen decreased exponentially. The fall-off in intensity is due tophotobleaching of the fluorescein molecules. The traces of beads withoutfluorescein molecules are used for background subtraction. Thedifference in the initial exponential decay between labeled andnonlabeled beads is integrated to give the total number of photoncounts, and this number is related to the number of molecules per bead.Therefore, it is possible to deduce the number of photons perfluorescein molecule that can be detected. This calculation indicatesthe radiation of about 40 to 50 photons per fluorescein molecule aredetected.

6. determination of the number of molecules per unit area

Aminopropylated glass microscope slides prepared according to themethods discussed above were utilized in order to establish the densityof labeling of the slides. The free amino termini of the slides werereacted with FITC (fluorescein isothiocyanate) which forms a covalentlinkage with the amino group. The slide is then scanned to count thenumber of fluorescent photons generated in a region which, using theestimated 40-50 photons per fluorescent molecule, enables thecalculation of the number of molecules which are on the surface per unitarea.

A slide with aminopropyl silane on its surface was immersed in a 1 mMsolution of FITC in DMF for 1 hour at about ambient temperature. Afterreaction, the slide was washed twice with DMF and then washed withethanol, water, and then ethanol again. It was then dried and stored inthe dark until it was ready to be examined.

Through the use of curves similar to those shown in FIG. 11 of Pirrunget al. (1992) U.S. Pat. No. 5,143,854, and by integrating thefluorescent counts under the exponentially decaying signal, the numberof free amino groups on the surface after derivitization was determined.It was determined that slides with labeling densities of 1 fluoresceinper 10³ ×10³ to .sup.˜ 2×2 nm could be reproducibly made as theconcentration of aminopropyltriethoxysilane varied from 10⁻⁵ % to 10⁻¹%.

7. removal of NVOC and attachment of a fluorescent marker

NVOC-GABA groups were attached as described above. The entire surface ofone slide was exposed to light so as to expose a free amino group at theend of the gamma amino butyric acid. This slide, and a duplicate whichwas not exposed, were then exposed to fluorescein isothiocyanate (FITC).

FIG. 12A of Pirrung et al. (1992) U.S. Pat. No. 5,143,854 illustratesthe slide which was not exposed to light, but which was exposed to FITC.The units of the x axis are time and the units of the y axis are counts.The trace contains a certain amount of background fluorescence. Theduplicate slide was exposed to 350 nm broadband illumination for about 1minute (12 mW/cm², .sup.˜ 350 nm illumination), washed and reacted withFITC. A large increase in the level of fluorescence is observed, whichindicates photolysis has exposed a number of amino groups on the surfaceof the slides for attachment of a fluorescent marker.

8. use of a mask in removal of NVOC

The next experiment was performed with a 0.1% aminopropylated slide.Light from a Hg--Xe arc lamp was imaged onto the substrate through alaser-ablated chrome-on-glass mask in direct contact with the substrate.

This slide was illuminated for approximately 5 minutes, with 12 mW of350 nm broadband light and then reacted with the 1 mM FITC solution. Itwas put on the laser detection scanning stage and a graph was plotted asa two-dimensional representation of position color-coded forfluorescence intensity. The experiment was repeated a number of timesthrough various masks. The fluorescence patterns for a 100×100 μm mask,a 50 μm mask, a 20 μm mask, and a 10 μm mask indicate that the maskpattern is distinct down to at least about 10 μm squares using thislithographic technique.

9. attachment of YGGFL and subsequent exposure to herz antibody and goatanti-mouse antibody

In order to establish that receptors to a particular polypeptidesequence would bind to a surface-bound peptide and be detected, Leuenkephalin was coupled to the surface and recognized by an antibody. Aslide was derivatized with 0.1% amino propyl-triethoxysilane andprotected with NVOC. A 500 μm checkerboard mask was used to expose theslide in a flow cell using backside contact printing. The Leu enkephalinsequence (H₂ N-tyrosine,glycine,glycine,phenylalanine,leucine-COOH,otherwise referred to herein as YGGFL) was attached via its carboxy endto the exposed amino groups on the surface of the slide. The peptide wasadded in DMF solution with the BOP/HOBT/DIEA coupling reagents andrecirculated through the flow cell for 2 hours at room temperature.

A first antibody, known as the Herz antibody, was applied to the surfaceof the slide for 45 minutes at 2 μg/ml in a supercocktail (containing 1%BSA and 1% ovalbumin also in this case). A second antibody, goatanti-mouse fluorescein conjugate, was then added at 2 μg/ml in thesupercocktail buffer, and allowed to incubate for 2 hours.

The results of this experiment were plotted as fluorescence intensity asa function of position. This image was taken at 10 μm steps and showedthat not only can deprotection be carried out in a well defined pattern,but also that (1) the method provided for successful coupling ofpeptides to the surface of the substrate, (2) the surface of a boundpeptide was available for binding with an antibody, and (3) thedetection apparatus capabilities were sufficient to detect binding of areceptor. Moreover, the Herz antibody is a sequence specific reagentwhich may be used advantageously as a sequence specific recognitionreagent. It may be used, if specificity is high, for sequencingpurposes, and, at least, for fingerprinting and mapping uses.

10. monomer-by-monomer formation of YGGFL and subsequent exposure tolabeled antibody

Monomer-by-monomer synthesis of YGGFL and GGFL in alternate squares wasperformed on a slide in a checkerboard pattern and the resulting slidewas exposed to the Herz antibody.

A slide is derivatized with the aminopropyl group, protected in thiscase with t-BOC (t-butoxycarbonyl). The slide was treated with TFA toremove the t-BOC protecting group. E-aminocaproic acid, which was t-BOCprotected at its amino group, was then coupled onto the aminopropylgroups. The aminocaproic acid serves as a spacer between the aminopropylgroup and the peptide to be synthesized. The amino end of the spacer wasdeprotected and coupled to NVOC-leucine. The entire slide was thenilluminated with 12 mW of 325 nm broadband illumination. The slide wasthen coupled with NVOCphenylalanine and washed. The entire slide wasagain illuminated, then coupled to NVOC-glycine and washed. The slidewas again illuminated and coupled to NVOC-glycine to form the sequenceshown in the last portion of FIG. 13A of Pirrung et al. (1992) U.S. Pat.No. 5,143,854.

Alternating regions of the slide were then illuminated using aprojection print using a 500×500 μm checkerboard mask; thus, the aminogroup of glycine was exposed only in the lighted areas. When the nextcoupling chemistry step was carried out, NVOC-tyrosine was added, and itcoupled only at those spots which had received illumination. The entireslide was then illuminated to remove all the NVOC groups, leaving acheckerboard of YGGFL in the lighted areas and in the other areas, GGFL.The Herz antibody (which recognizes the YGGFL, but not GGFL) was thenadded, followed by goat anti-mouse fluorescein conjugate.

The resulting fluorescence scan showed dark areas containing thetetrapeptide GGFL, which is not recognized by the Herz antibody (andthus there is no binding of the goat anti-mouse antibody withfluorescein conjugate), and red areas in which YGGFL was present. TheYGGFL pentapeptide is recognized by the Herz antibody and, therefore,there is antibody in the lighted regions for the fluorescein-conjugatedgoat anti-mouse to recognize.

Similar patterns for a 50 μm mask used in direct contact ("proximityprint") with the substrate provided a pattern which was more distinctand the corners of the checkerboard pattern were touching as a result ofthe mask being placed in direct contact with the substrate (whichreflects the increase in resolution using this technique).

11. monomer-by-monomer synthesis of YGGFL and PGGFL

A synthesis using a 50 μm checkerboard mask was conducted. However, Pwas added to the GGFL sites on the substrate through an additionalcoupling step. P was added by exposing protected GGFL to light through amask, and subsequence exposure to P in the manner set forth above.Therefore, half of the regions on the substrate contained YGGFL and theremaining half contained PGGFL.

The fluorescence plot for this experiment showed the regions are againreadily discernable between those in which binding did and did notoccur. This experiment demonstrated that antibodies are able torecognize a specific sequence and that the recognition is notlength-dependent.

12. monomer-by-monomer synthesis of YGGFL and YPGGFL

In order to further demonstrate the operability of the invention, a 50μm checkerboard pattern of alternating YGGFL and YPGGFL was synthesizedon a substrate using techniques like those set forth above. Theresulting fluorescence plot showed that the antibody was clearly able torecognize the YGGFL sequence and did not bind significantly at theYPGGFL regions.

13. synthesis of an array of sixteen different amino acid sequences andestimation of relative binding affinity to herz antibody

Using techniques similar to those set forth above, an array of 16different amino acid sequences (replicated four times) was synthesizedon each of two glass substrates. The sequences were synthesized byattaching the sequence NVOC-GFL across the entire surface of the slides.Using a series of masks, two layers of amino acids were then selectivelyapplied to the substrate. Each region had dimensions of 0.25 cm×0.0625cm. The first slide contained amino acid sequences containing only L-amino acids while the second slide contained selected D- amino acids.Various regions on the first and second slides, were duplicated fourtimes on each slide. The slides were then exposed to the Herz antibodyand fluorescein-labeled goat anti-mouse antibodies.

A fluorescence plot of the first slide, which contained only L- aminoacids showed red areas (indicating strong binding, i.e., 149,000 countsor more) and black areas (indicating little or no binding of the Herzantibody, i.e., 20,000 counts or less). The sequence YGGFL was clearlymost strongly recognized. The sequences YAGFL and YSGFL also exhibitedstrong recognition of the antibody. By contrast, most of the remainingsequences showed little or no binding. The four duplicate portions ofthe slide were extremely consistent in the amount of binding showntherein.

A fluorescence plot of the D- amino acid slide indicated that strongestbinding was exhibited by the YGGFL sequence. Significant binding wasalso detected to YaGFL, YsGFL, and YpGFL. The remaining sequences showedless binding with the antibody. Low binding efficiency of the sequenceyGGFL was observed.

Table 6 lists the various sequences tested in order of relativefluorescence, which provides information regarding relative bindingaffinity.

                  TABLE 6    ______________________________________    Apparent Binding to Herz Ab           L- a.a. Set                  D- a.a. Set    ______________________________________           YGGFL  YGGFL           YAGFL  YaGFL           YSGFL  YsGFL           LGGFL  YpGFL           FGGFL  fGGFL           YPGFL  yGGFL           LAGFL  faGFL           FAGFL  wGGFL           WGGFL  yaGFL                  fpGFL                  waGFL    ______________________________________

14. illustrative alternative embodiment

According to an alternative embodiment of the invention, the methodsprovide for attaching to the surface a caged binding member which, inits caged form, has a relatively low affinity for other potentiallybinding species, such as receptors and specific binding substances. Suchtechniques are more fully described in copending application Ser. No.404,920, filed Sep. 8, 1989, and incorporated herein by reference forall purposes. See also Ser. No. 07/435,316, now abandoned, and Barrettet al. (1993) U.S. Pat. No. 5,252,743, each of which is herebyincorporated herein by reference.

According to this alternative embodiment, the invention provides methodsfor forming predefined regions on a surface of a solid support, whereinthe predefined regions are capable of immobilizing receptors. Themethods make use of caged binding members attached to the surface toenable selective activation of the predefined regions. The caged bindingmembers are liberated to act as binding members ultimately capable ofbinding receptors upon selective activation of the predefined regions.The activated binding members are then used to immobilize specificmolecules such as receptors on the predefined region of the surface. Theabove procedure is repeated at the same or different sites on thesurface so as to provide a surface prepared with a plurality of regionson the surface containing, for example, the same or different receptors.When receptors immobilized in this way have a differential affinity forone or more ligands, screenings and assays for the ligands can beconducted in the regions of the surface containing the receptors.

The alternative embodiment may make use of novel caged binding membersattached to the substrate. Caged (unactivated) members have a relativelylow affinity for receptors of substances that specifically bind touncaged binding members when compared with the corresponding affinitiesof activated binding members. Thus, the binding members are protectedfrom reaction until a suitable source of energy is applied to theregions of the surface desired to be activated. Upon application of asuitable energy source, the caging groups labilize, thereby presentingthe activated binding member. A typical energy source will be light.

Once the binding members on the surface are activated they may beattached to a receptor. The receptor chosen may be a monoclonalantibody, a nucleic acid sequence, a drug receptor, etc. The receptorwill usually, though not always, be prepared so as to permit attachingit, directly or indirectly, to a binding member. For example, a specificbinding substance having a strong binding affinity for the bindingmember and a strong affinity for the receptor or a conjugate of thereceptor may be used to act as a bridge between binding members andreceptors if desired. The method uses a receptor prepared such that thereceptor retains its activity toward a particular ligand.

Preferably, the caged binding member attached to the solid substratewill be a photoactivatable biotin complex, i.e., a biotin molecule thathas been chemically modified with photoactivatable protecting groups sothat it has a significantly reduced binding affinity for avidin oravidin analogs than does natural biotin. In a preferred embodiment, theprotecting groups localized in a predefined region of the surface willbe removed upon application of a suitable source of radiation to givebinding members, that is biotin or a functionally analogous compoundhaving substantially the same binding affinity for avidin or avidinanalogs as does biotin.

In another preferred embodiment, avidin or an avidin analog is incubatedwith activated binding members on the surface until the avidin bindsstrongly to the binding members. The avidin so immobilized on predefinedregions of the surface can then be incubated with a desired receptor orconjugate of a desired receptor. The receptor will preferably bebiotinylated, e.g., a biotinylated antibody, when avidin is immobilizedon the predefined regions of the surface. Alternatively, a preferredembodiment will present an avidin/biotinylated receptor complex, whichhas been previously prepared, to activated binding members on thesurface.

II. FINGERPRINTING

The above section on generation of reagents for sequencing providesspecific reagents useful for fingerprinting applications. Fingerprintingembodiments may be applied towards polynucleotide fingerprinting,polypeptide fingerprinting, cell and tissue classification, cell andtissue temporal development stage classification, diagnostic tests,forensic uses for individual identification, classification oforganisms, and genetic screening of individuals. Mapping applicationsare also described below.

A. Polynucleotide Fingerprint

Polynucleotide fingerprinting may use reagents similar to thosedescribed above for probing a sequence for the presence of specificsubsequences found therein. Typically, the subsequences used forfingerprinting will be longer than the sequences used in oligonucleotidesequencing. In particular, specific long segments may be used todetermine the similarity of different samples of nucleic acids. They mayalso be used to fingerprint whether specific combinations of informationare provided therein. Particular probe sequences are selected andattached in a positional manner to a substrate. The means for attachmentmay be either using a caged biotin method described, e.g., in Barrett etal. (1993) U.S. Pat. No. 5,242,743, or by another method using targetingmolecules. For example, a short polypeptide of specific sequence may beattached to an oligonucleotide and targeted to specific positions on asubstrate having antibodies attached thereto, the antibodies exhibitingspecificity for binding to those short peptide sequences. In anotherembodiment, an unnatural nucleotide or similar complementary bindingmolecule may be attached to the fingerprinting probe and the probethereby directed towards complementary sequences on a VLSIPS substrate.Typically, unnatural nucleotides would be preferred, e.g., unnaturaloptical isomers, which would not interfere with natural nucleotideinteractions.

Having produced a substrate with particular fingerprint probes attachedthereto at positionally defined regions, the substrate may be used in amanner quite similar to the sequencing embodiment to provide informationas to whether the fingerprint probes are detecting the correspondingsequence in a target sequence. This will often provide informationsimilar to a Southern blot hybridization.

B. Polypeptide Fingerprint

A polypeptide fingerprint may be performed using antibodies whichrecognize specific antigens on the polypeptide. For example, monoclonalantibodies which recognize specific sequences or antigens on apolypeptide may be used to determine whether those epitopes are found ona particular protein. For example, particular patterns of epitopes wouldbe found on various types of proteins. This will lead to the discoverythat specific epitopes, or antigenic determinants, which arecharacteristic of, e.g., beta sheet segments, will be identified as willparticular different types of domains in various protein types. Thus, ascreening method may be devised which can classify polypeptides, eithernative or denatured, into various new classes defined by the epitopesexisting thereon.

In addition, once the substrate is generated in the manners describedabove, a target peptide is exposed to the substrate. The target may beeither native or denatured, though the conditions used to denature thepolypeptide may interfere with the specific interaction between thepolypeptide and the recognition reagent. This method is not dependent onthe fact that the polypeptide is a single chain, thus protein complexesmay also be fingerprinted using this methodology. Structures such asmulti-subunit proteins, associations of proteins, ribosomes,nucleosomes, and other small cellular structures may also befingerprinted and classified according to the presence of specificrecognizable features thereon.

Peptide fingerprinting may be useful, for example, in correlating withparticular physiological conditions or developmental stages of a cell ororganism. Thus, a biological sample may be fingerprinted to determinethe presence in that sample of a plurality of different polypeptideswhich are each individually fingerprinted. In an alternative embodiment,a polypeptide itself is not fingerprinted but a biological sample isfingerprinted searching for specific epitopes, e.g., polypeptide,carbohydrate, nucleic acid, or any of a number of other specificrecognizable structural features.

The conditions for the interactions using antibodies is described, e.g.,in Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold SpringHarbor Press, New York. The conditions should be titrated fortemperature, buffer composition, time, and other important parameters inan antibody interaction.

C. Cell Classification Scheme

The present invention can be used for cell classification usingfingerprinting type technology as described above in the polypeptidefingerprint. Classes of cells are typically defined by the presence ofcommon functions which are usually reflected by structural features.Thus, a plant cell is classified differently from an animal cell by anumber of structural features. Given an unknown cell, the presentinvention provides improved means for distinguishing the different celltypes. Once a cell classification scheme is developed and the structuralfeatures which define it are identified using the present invention,homogeneous cell population expressing these features may be separatedfrom others. Standard cell sorters may be coupled with recognitionreagents and labels which can distinguish various cell types.

a. T-Cell Classes

T-cell classes are defined on the basis of expression of particularantigens characteristic of each class. For example, mouse T-celldifferentiation markers include the LY antigens. With the plurality ofdifferent antigens which may be tested using antibody or otherrecognition reagents, new populations and classes of cells may bedefined. For example, different neural cell types may be defined on thebasis of cell surface antigens. Different tissue types will be definedon the basis of tissue specific antigens. Developmental cell classeswill be similarly defined. All of these screenings can make use of theVLSIPS substrates with specific recognition molecules attached thereto.The substrates are exposed to the cell types directly, assaying forattachment of cells to specific regions, or are exposed to products of apopulation of cells, e.g., a supernatant, or a cell lysate.

Once a cell classification scheme has been correlated with specificstructural markers therein, reagents which recognize those features maybe developed and used in a fluorescence activated cell sorter asdescribed, e.g., in Dangl, J. and Herzenberg (1982) J. ImmunologicalMethods 52: 1-14; and Becton Dickinson, Fluorescence Activated CellSorters Division, San Jose, Calif. This will provide a homogeneouspopulation of cells whose function has been defined by structure.

b. B-Cell Classes

The present cell classification scheme may also be used to determinespecific B-cell classes. For example, B-cells specific for producingIgM, IgG, IgD, IgE, and IgA may be defined by the internal expression ofspecific mRNA sequences encoding each type of immunoglobulin. Theclassification scheme may depend on either extracellularly expressedmarkers which are correlated as being diagnostic of specific stages indevelopment, or intracellular mRNA sequences which indicate particularfunctions.

D. Temporal Development Scheme

1. Developmental Antigens

The present fingerprinting invention also allows cell classification byexpression of developmental antigens. For example, a lymphocyte stemcell expresses a particular combination of antigens. As the lymphocytedevelops through a program developmental scheme, at various stages itexpresses particular antigens which are diagnostic of particular stagesin development. Again, the fingerprinting methodology allows for thedefinition of specific structural features which are diagnostic ofdevelopmental or functional features which will allow classification ofcells into temporal developmental classes. Cells, products of thosecells, or lysates of those cells will be assayed to determine thedevelopmental stage of the source cells. In this manner, once adevelopmental stage is defined, specific synchronized populations ofcells will be selected out of another population. These synchronizedpopulations may be very important in determining the biologicalmechanisms of development.

2. Developmental mRNA Expression

Besides expressed antigens, the present invention also allows forfingerprinting of the mRNA population of a cell. In this fashion, themRNA population, which should be a good determinant of developmentalstage, will be correlated with other structural features of the cell. Inthis manner, cells at specific developmental stages will becharacterized by the intracellular environment, as well as theextracellular environment. The present invention also allows thecombination of definitions based, in part, upon antigens and, in part,upon mRNA expression.

In one embodiment, the two may be combined in a single incubation step.A particular incubation condition may be found which is compatible withboth hybridization recognition and non-hybridization recognitionmolecules. Thus, e.g., an incubation condition may be selected whichallows both specificity of antibody binding and specificity of nucleicacid hybridization. This allows simultaneous performance of both typesof interactions on a single matrix. Again, where developmental mRNApatterns are correlated with structural features, or with probes whichare able to hybridize to intracellular mRNA populations, a cell sortermay be used to sort specifically those cells having desired mRNApopulation patterns.

E. Diagnostic Tests

The present invention also provides the ability to perform diagnostictests. Diagnostic tests typically are based upon a fingerprint typeassay, which tests for the presence of specific diagnostic structuralfeatures. Thus, the present invention provides means for viral strainidentification, bacterial strain identification, and other diagnostictests using positionally defined specific reagents. The presentinvention also allows for determining a spectrum of allergies,diagnosing a biological sample for any or all of the above, and testingfor many other conditions.

1. Viral Identification

The present invention provides reagents and methodology for identifyingviral strains. The specific reagents may be either antibodies orrecognition proteins which bind to specific viral epitopes preferablysurface exposed, but may make use of internal epitopes, e.g., in adenatured viral sample. In an alternative embodiment, the viral genomemay be probed for specific sequences which are characteristic ofparticular viral strains. As above, a combination of the two may beperformed simultaneously in a single interaction step, or in separatetests, e.g., for both genetic characteristics and epitopecharacteristics.

2. Bacterial Identification

Similar techniques will be applicable to identifying a bacterial source.This may be useful in diagnosing bacterial infections, or in classifyingsources of particular bacterial species. For example, the bacterialassay may be useful in determining the natural range of survivability ofparticular strains of bacteria across regions of the country or indifferent ecological niches.

3. Other Microbiological Identifications

The present invention provides means for diagnosis of othermicrobiological and other species, e.g., protozoal species and parasiticspecies in a biological sample, but also provides the means for assayinga combination of different infections. For example, a biologicalspecimen may be assayed for the presence of any or all of thesemicrobiological species. In human diagnostic uses, typical samples willbe blood, sputum, stool, urine, or other samples.

4. Allergy Tests

An immobilized set of antigens may be attached to a solid substrate and,instead of the standard skin reaction tests, a blood sample may beassayed on such a substrate to determine the presence of antibodies,e.g., IgE or other type antibodies, which may be diagnostic of anallergic or immunological susceptibility. A standard radioallergosorbenttest (RAST) may be used to check a much larger population of antigens.

In addition, an allergy like test may be used to diagnose theimmunological history of a particular individual. For example, bytesting the circulating antibodies in a blood sample, which reflects theimmunological history and memory of an individual, it may be determinedwhat infections may not have been historically presented to the immunesystem. In this manner, it may be possible to specifically supplement animmune system for a short period of time with IgG fractions made up ofspecific types of gamma globulins. Thus, hepatitis gamma globulininjections may be better designed for a particular environment to whicha person is expected to be exposed. This also provides the ability toidentify genetically equivalent individuals who have immunologicallydifferent experiences. Thus, a blood sample from an individual who has aparticular combination of circulating antibodies will likely bedifferent from the combination of circulating antibodies found in agenetically similar or identical individual. This could allow for thedistinction between clones of particular animals, e.g., mice, rats, orother animals.

F. Individual Identification

The present invention provides the ability to fingerprint and identify agenetic individual. This individual may be a bacterial or lowermicroorganism, as described above in diagnostic tests, or of a plant oranimal. An individual may be identified genetically or immunologically,as described.

1. Genetic

Genetic fingerprinting has been utilized in comparing different relatedspecies in Southern hybridization blots. Genetic fingerprinting has alsobeen used in forensic studies, see, e.g., Morris et al. (1989) J.Forensic Science 34: 1311-1317, and references cited therein. Asdescribed above, an individual may be identified genetically by asufficiently large number of probes. The likelihood that anotherindividual would have an identical pattern over a sufficiently largenumber of probes may be statistically negligible. However, it is oftenquite important that a large number of probes be used where thestatistical probability of matching is desired to be particularly low.In fact, the probes will optimally be selected for having highheterogeneity among the population. In addition, the fingerprint methodmay make use of the pattern of homologies indicated by a series of moreand more stringent washes. Then, each position has both a sequencespecificity and a homology measurement, the combination of which greatlyincreases the number of dimensions and the statistical likelihood of aperfect pattern match with another genetic individual.

2. Immunological

As indicated above in the diagnostic tests, it is possible to identify aparticular immune system within a genetically homogeneous class oforganisms by virtue of their immunological history. For example, a largecolony of cloned mice may be distinguishable by virtue of eachimmunological history. For example, one mouse may have had animmunological response to exposure to antigen A to which her geneticallyidentical sibling may have not been exposed. By virtue of thisdifferential history, the first of the pair will likely have a highantibody titer against the antigen A whereas her genetically identicalsibling will have not had a response to that antigen by virtue of neverhaving been exposed to it. For this reason, immune systems may beidentified by their immunological memories. Thus, immunologicalexperience may also be a means for identifying a particular individualat a particular moment in her lifetime.

This same immunological screening may be used for other sorts ofidentifiable biological products. For example, an individual may beidentified by her combination of expressed proteins. These proteins mayreflect a physiological state of the individual, and would thus beuseful in certain circumstances where diagnostic tests may be performed.For example, an individual may be identified, in part, by the presenceof particular metabolic products.

In fact, a plant origin may be determined by virtue of having within itsgenome an unnatural sequence introduced to it by genetic breeders. Thus,a marker nucleic acid sequence may be introduced as a means to determinewhether a genetic strain of a plant or animal originated from anotherparticular source.

G. Genetic Screening

1. test alleles with markers

The present invention provides for the ability to screen for geneticvariations of individuals. For example, a number of genetic diseases arelinked with specific alleles. See, e.g., Scriber, C. et al. (eds.)(1989) The Metabolic Bases of Inherited Disease, McGraw-Hill, New York.In one embodiment, cystic fibrosis has been correlated with a specificgene, see, Gregory et al. (1990) Nature 347: 382-386. A number ofalleles are correlated with specific genetic deficiencies. See, e.g.,McKusick, V. (1990) Genetic Inheritance in Man: Catalogs of AutosomalDominant, Autosomal Recessive, and X-linked Phenotypes, Johns HopkinsUniversity Press, Baltimore; Ott, J. (1985) Analysis of Human GeneticLinkage, Johns Hopkins University Press, Baltimore; Track, R. et al.(1989) Banbury Report 32: DNA Technology and Forensic Science, ColdSpring Harbor Press, New York; each of which is hereby incorporatedherein by reference.

2. Amniocentesis

Typically, amniocentesis is used to determine whether chromosometranslocations have occurred. The mapping procedure may provide the means for determining whether these translocations have occurred, and fordetecting particular alleles of various markers.

III. MAPPING

A. Positionally Located Clones

The present invention allows for the positional location of specificclones useful for mapping. For example, caged biotin may be used forspecifically positioning a probe to a location on a matrix pattern.

In addition, the specific probes may be positionally directed tospecific locations on a substrate by targeting. For example, polypeptidespecific recognition reagents may be attached to oligonucleotidesequences which can be complementarily targeted to specific locations ona VLSIPS™ Technology substrate. Hybridization conditions, as applied foroligonucleotide probes, will be used to target the reagents to locationson a substrate having complementary oligonucleotides synthesizedthereon. In another embodiment, oligonucleotide probes may be attachedto specific polypeptide targeting reagents such as an antigen orantibody. These reagents can be directed towards a complementary antigenor antibody already attached to a VLSIPS substrate.

In another embodiment, an unnatural nucleotide which does not interferewith natural nucleotide complementary hybridization may be used totarget oligonucleotides to particular positions on a substrate.Unnatural optical isomers of natural nucleotides should be idealcandidates.

In this way, short probes may be used to determine the mapping of longtargets or long targets may be used to map the position of shorterprobes. See, e.g., Craig et al. 1990 Nuc. Acids Res. 18: 2653-2660.

B. Positionally Defined Clones

Positionally defined clones may be transferred to a new substrate byeither physical transfer or by synthetic means. Synthetic means mayinvolve either a production of the probe on the substrate using theVLSIPS™ Technology synthetic methods, or may involve the attachment of atargeting sequence made by VLSIPS synthetic methods which will targetthat positionally defined clone to a position on a new substrate. Bothmethods will provide a substrate having a number of positionally definedprobes useful in mapping.

IX. Conclusion

The present inventions provide greatly improved methods and apparatusfor synthesis of polymers on substrates. It is to be understood that theabove description is intended to be illustrative and not restrictive.Many embodiments will be apparent to those of skill in the art uponreviewing the above description. By way of example, the invention hasbeen described primarily with reference to the use of photoremovableprotective groups, but it will be readily recognized by those of skillin the art that sources of radiation other than light could also beused. For example, in some embodiments it may be desirable to useprotective groups which are sensitive to electron beam irradiation,x-ray irradiation, in combination with electron beam lithograph, orx-ray lithography techniques. Alternatively, the group could be removedby exposure to an electric current. The scope of the invention should,therefore, be determined not with reference to the above description,but should instead be determined with reference to the appended claims,along with the full scope of equivalents to which such claims areentitled.

All publications and patent applications referred to herein areincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyincorporated by reference. The present invention now being fullydescribed, it will be apparent to one of ordinary skill in the art thatmany changes and modifications can be made thereto without departingfrom the spirit or scope of the appended claims.

What is claimed is:
 1. A method of identifying nucleotide differencesbetween the sequence of a target nucleic acid and the sequence of areference nucleic acid comprising:a) providing a substrate having atleast 1000 different polynucleotide probes of known sequence at knownlocations, attached at a density of at least 10,000 probes per squarecm; b) contacting the target nucleic acid with the polynucleotide probesattached to the substrate under conditions for high specificitycomplementary hybridization; c) determining which polynucleotide probeshave hybridized with the target nucleic acid; and d) using a computer to(i) compare the sequence of the reference nucleic acid with thesequences of the polynucleotide probes that have hybridized with thetarget nucleic acid and (ii) identify the nucleotide differences betweenthe sequence of the target nucleic acid and the sequence of thereference nucleic acid.
 2. the method of claim 1 wherein the substrateis produced by the process of:i) providing a substrate having sitesprotected with photosensitive protective groups; ii) selectivelyirradiating portions of the substrate with light to remove saidphotoprotective groups and expose said sites; iii) attaching anucleotide to the site, said nucleotide protected with a photosensitiveprotective group; and iv) repeating steps ii) and iii) so as to attachto the substrate at least 1000 different polynucleotide probes of knownsequence at known locations.
 3. The method of claim 1 further comprisinglabeling the target nucleic acid with a fluorescent label.
 4. The methodof claim 3 wherein the polynucleotide probes that have hybridized withthe target nucleic acid are determined by detecting the fluorescentlabel.
 5. The method of claim 1 wherein the density of different probesof known sequence is at least 100,000 per square centimeter.
 6. Themethod of claim 1 wherein the density of different probes of knownsequence is at least 1,000,000 per square centimeter.